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New competitive antagonistic peptides to MC-1R as whitening agents
November, 2009
 

Pigmentation of human skin is closely involved in protection against external stresses, in particular exposure to
UV radiation. The skin is the main barrier to the environmental stresses, and relies on melanocytes to provide
photoprotection and thermoregulation by synthesizing melanin. The color of the skin is largely determined by
melanocytes and melanin is the main contributor to pigmentation.


In some areas, especially in Asia, lighter skin color is considered as a symbol of beauty and therefore there
are continuous search for the new and powerful whitening agents. Whitening agents currently found in the
market such as antioxidants and tyrosinase inhibitors show numerous side effects such as irritation,
sensitization, cytotoxicity, and instability.

With the efforts to search for more effective and powerful antagonists for the melanocortin-1 receptor, we
have found several candidates with good in vitro profiles and stabilities. As shown below in in vitro efficacy
data, we have tested four different peptide candidates, two tripeptides, W310 and W320 and two
tetrapeptides, W410 and W420.

1. Inhibition of melanogenesis in vitro


Melanoma B16F10 cells were pre-treated with various concentrations of peptide samples for 30 minutes and
then stimulated with or without 1ppm α-MSH. After 48 h, the cells were washed with PBS and dissolved in
100
2N NaOH containing 50% DMSO at 60°C and absorbance was measured at 475nm using an ELISA
reader.


2. Inhibition of tyrosinase activity in vitro

Tyrosinase activity was examined in melanoma B16F10 cells. After treatment with various concentrations of
peptide samples, the cells were stimulated with or without 1ppm α-MSH. After 72 hours, cells were removed
from culture dishes and washed with PBS. Then, the cells were lysed and the L-Dopa oxidation activity of
tyrosinase was measured at 492 nm by spectrophotometer.






3. Inhibition of cellular cAMP levels in vitro

 

To examine the inhibitory effect of tri- and tetrapeptides on cAMP activation, the intracellular level of cAMP
was measured by direct ELISA. Melanoma B16F10 cells were exposed to peptide samples for 20 min and
were lysed. The intracellular cAMP was measured at 405nm by spectrophotometer.




4. Signaling Study

 

To determine whether tri- and tetrapeptides are MC1R-specific antagonists, Melanoma B16F10 cells were
activated with several melanogenenic stimulators.
α-MSH, Forskolin, 8-bromo-cAMP, PMA, and 8-bromo-cGMP
were used for activating MC1R, adenyl cyclase, cAMP, PKC and PKG respectively. After 48 h of incubation
with
W320, W410 and W420, the melanin contents were measured by an ELISA reader. 

 


As shown in the graph above, two tetrapeptides, W410 and W420 effectively inhibited the melanin synthesis
that stimulated by
α-MSH. Both peptides also lowered the melanin contents that stimulated by PMA, which
means that both peptides may be involved in PKC-related signaling.


5. Down-regulation of melanogenic genes by W320 and W410 in vitro

 

Melanoma B16F10 cells were treated with various concentrations of W320 or W410, and then stimulated with
or without 1ppm of
α-MSH. After 48 h, total cellular RNA was extracted and reverse-transcription PCR was
performed to determine tyrosinase, TRP1 and TRP2. β
-Actin mRNA level was used for sample standardization.
W320 and W410 significantly down-regulated the expression of melanogenic genes.



[Modification of tetrapeptide W410 with two different fatty acids]

 

The most effective antagonistic peptide among four different peptides, W410 was modified with two different
fatty acids, palmitic or myristic acid and the activities of both modified tetrapeptides, W411 and W412 were
compared to the original unmodified tetrapeptide, W410. 

6. Inhibition of melanogenesis by W411 and W412 in vitro

 

Melanoma B16F10 cells were pre-treated with various concentrations of W410 and its fatty acid derivatives
(W411 and W412) for 30 minutes and then stimulated with or without 1ppm
α-MSH. After 48 h, the cells were
washed with PBS and dissolved
in 100 2N NaOH containing 50% DMSO at 60°C and absorbance was
measured at 475nm using an ELISA reader. As shown below, W411 and W412, the fatty acid derivatives of
W410 more effectively inhibited the melanin synthesis than unmodified tetrapeptide W410.




7. Inhibition of tyrosinase activity by W411 and W412 in vitro

 

Tyrosinase activity was examined in melanoma B16F10 cells. After treatment with various concentrations of
W410 and its fatty acid derivatives (W411 and W412), the cells were stimulated with of without 1ppm of
α-MSH. After 72 hours, cells were removed from culture dishes and washed with PBS. Then, the cells were
lysed and the L-Dopa oxidation activity of tyrosinase was measured at 492 nm by spectrophotomet
er. Again as
shown below, W411 and W412, the fatty acid derivatives of W410 more effectively inhibited the melanin
synthesis than unmodified tetrapeptide W410.


 

8. Inhibition of melanogenic genes by W411 and W412 in vitro

 

Melanoma B16F10 cells were pre-treated with various concentrations of W320, W410 or its fatty acid derivatives
(W411 and W412) and then stimulated with or without 1ppm of
α-MSH. After 48 h, total cellular RNA was
extracted and reverse-transcription PCR was performed to determine tyrosinase, TRP1 and TRP2. β
-Actin mRNA
level was used for sample standardization.
 The fatty acid derivatives of W410 are far more effective than W320
and W410.







[Conclusions]

 In conclusion, a tetrapeptide modified with myristic acid was found to be the most effective MC-1R
 antagonistic peptide among the peptides tested. Additional studies on this peptide are currently underway.