Inhibition of Sik causes pathways of melaningenisis
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Uhohinc
Jun 13, 2017, 2:58:53 PM6/13/17
to Clinuvel Afamelanotide Scenesse Vitiligo Porphyria CUV ASX.CUV CLVLY ur9
Uhohinc
Mar 15, 2021, 2:13:33 AM3/15/21
to Clinuvel Afamelanotide SCENESSE senescence CUV ASX.CUV CLVLY ur9
Credit to ? on sharetease..........note David Fisher from Harvard.........gives credability here to some incredulous claims........
On Tuesday, June 13, 2017 at 11:58:53 AM UTC-7 Uhohinc wrote:
http://www.smithsonianmag.com/innovation/real-tan-no-sun-needed-180963667/
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Mar 15, 2021, 6:36:14 AM3/15/21
to Clinuvel Afamelanotide SCENESSE senescence CUV ASX.CUV CLVLY ur9
topical SIK Inhibitors Induce Human Skin
Eumelanization
Treatment of human skin explants with passive topical applica-
tion of the second-generation SIK inhibitors, YKL 06-061 and
YKL 06-062, induced significant pigmentation after 8 days of
treatment (13/day), but no significant gross pigmentation was
observed in skin treated with HG 9-91-01 (Figure 4A). Fontana-
Masson staining revealed increased melanin content in skin
treated with YKL 06-061 or YKL 06-062 and marginally increased
melanin in skin treated with HG 9-91-01, as compared with con-
trol (Figure 4B). This effect was reproducible with independent
preparations of synthesized drugs applied passively (via pipette)
to the top of different human skin explants (Figure 4C and 4D).
Mechanical application of the first-generation SIK inhibitor HG
9-91-01, by rubbing via an applicator, induced significant gross
pigmentation (Figure 4E), and increased melanin content was
observed upon Fontana-Masson staining of skin sections (Fig-
ure 4F), suggesting that HG 9-91-01’s limited human skin pene-
tration can be, at least partially, overcome through mechanical
application. YKL 06-061 and YKL 06-062 did not require
2180 Cell Reports 19, 2177–2184, June 13, 2017
mechanical application (rubbing) to induce significant human
epidermal darkening.
DISCUSSION
These results illustrate the development and successful applica-
tion of small-molecule SIK inhibitors for topical induction of skin
pigmentation independently of UV irradiation in human skin. SIK
inhibitors were shown to induce enhanced expression of the
MITF transcription factor, which is known to regulate expression
of numerous pigment enzymes that promote biosynthesis of eu-
melanin. A new generation of SIK inhibitors was developed,
based on strategies for enhancing the likelihood of skin penetra-
tion through optimizing molecular size and lipophilicity. Two
such SIK-targeted inhibitors, YKL 06-061 and YKL 06-062,
were shown to induce similar responses both in vitro and when
applied to human skin explants. In addition to upregulating
mRNA levels of MITF and TRPM1, topical SIK inhibitors were
seen to trigger the transfer of melanosomes into epidermal
keratinocytes in a manner that recapitulates the perinuclear
capping (subcellular localization) seen in normal human
AB
C
D
E
F
Figure 4. Treatment of Human Skin Ex-
plants with 37.5 mM of SIK Inhibitor Induces
Pigmentation
(A) Human breast skin explants treated with pas-
sive application of vehicle control (70% ethanol,
30% propylene glycol) or 37.5 mM SIK inhibitor
YKL 06-061, YKL 06-062, or HG 9-91-01 for 8 days
(10 mL; 13/day). Image was taken 2 days after the
end of treatment (image is representative of two of
n = 3 experiments).
(B) Fontana-Masson (top panel) and H&E (bottom
panel) staining (magnification, 4003) of breast skin
described in (A). Scale bar represents 25 mm.
(C) Human breast skin explants treated with pas-
sive application of vehicle control or 37.5 mM SIK
inhibitor YKL 06-061, YKL 06-062, or HG 9-91-01
for 5 days (10 mL; 23/day). Image was taken 1 day
after the end of treatment (image is representative
of n = 1 experiment).
(D) Human breast skin explants treated with pas-
sive application of vehicle control or 37.5 mM SIK
inhibitor YKL 06-061, YKL 06-062, or HG 9-91-01
for 6 days (10 mL; 23/day). Image was taken 1 day
after the end of treatment (image is representative
of n = 1 experiment).
(E) Human breast skin explants treated with me-
chanical application of vehicle control or 50 mM
(50 mL for 1 day; 13/day) or 25 mM (50 mL for
3 days; 33/day) HG 9-91-01. Image was taken
4 days after the start of treatment (image is
representative of n = 1 experiment).
(F) Fontana-Masson (top panels) and H&E (bottom
panels) staining (magnification, 4003) of human
skin explants described in (E). Scale bar repre-
sents 25 mm.
epidermal pigmentation. Thus, SIK-inhib-
itor treatments appear to induce not only
synthesis of melanin but also melanoso-
mal maturation, export, and localization
features, even after import into keratinocytes. These features
closely resemble the previously observed behavior of forskolin
treatment in red-haired mice (D’Orazio et al., 2006).
Topical application of small-molecule, UV-independent
pigment inducers has not yet been examined in humans and
would require careful considerations of safety. For example,
the induction of dark pigmentation is associated with the lowest
risk of most skin cancers in humans (Armstrong and Kricker,
2001; Pennello et al., 2000), and this pigment synthesis is
believed to be dependent upon MITF (Bertolotto et al., 1998).
However, fixed genomic mutation or amplification of the MITF
gene can be oncogenic in certain contexts (Bertolotto et al.,
2011; Garraway et al., 2005; Yokoyama et al., 2011). Reversible
upregulation of MITF, as reported here, is also likely to occur in
routine instances of UV tanning, and constitutive elevation of
MITF is likely in the skin of individuals with darker pigmentation
levels; neither would be anticipated to trigger genomic mutation
of the MITF gene. Analogously, transient administration of re-
combinant hematopoietic growth factors has not been associ-
ated with formation of oncogenic transformation or leukemia
(Dombret et al., 1995; Ohno et al., 1990). In mice, topical
Cell Reports 19, 2177–2184, June 13, 2017 2181
forskolin’s pigmentary rescue in ‘‘redheads’’ resulted in signifi-
cant protection from UV carcinogenesis, without apparent asso-
ciated toxicities over many months of treatment (D’Orazio et al.,
2006). A recent study has utilized injections of the synthetic
alpha-MSH analog, afamelanotide, for treatment of photosensi-
tivity associated with erythropoietic protoporphyria. Pigmented
lesions/melanoma were carefully evaluated and reported not to
occur at elevated risk (Langendonk et al., 2015).
Our in vivo studies demonstrate that topical SIK inhibitor can
be applied with localized SIK inhibition and no detected systemic
effects in mice (such as failure to thrive); and although it has been
previously shown that SIK1 inhibition leads to cell-cycle arrest in
epithelial cells, this was dependent on the presence of trans-
forming growth factor b(TGF-b)(Lo
¨nn et al., 2012), and we
observed normal skin turnover with no morphological changes
of the skin (measured grossly or histologically, other than
pigment/color). During normal UV-induced tanning, MC1R acti-
vation leads to enhanced PKA activity (Newton et al., 2005),
and PKA-dependent phosphorylation of SIK1 (Takemori et al.,
2002), SIK2 (Horike et al., 2003), and SIK3 (Katoh et al., 2006) de-
creases their kinase activity. Since our compounds’ activity is
analogous to the on/off switch of UV-induced tanning, we
believe that it will be a safe, viable method of topical pigment
production, though it may be important to assure localized deliv-
ery to skin.
The half-life of melanin in skin is thought to be several weeks
and diminishes primarily after superficial keratinocyte sloughing.
Most epidermal melanin resides within keratinocytes after trans-
fer of melanosomes from melanocytes. Therefore, it is possible
that small-molecule approaches like that described here might
be achievable, or maintained, through intermittent pulse-dosing
strategies, thereby further limiting systemic drug exposure.
In conclusion, these studies describe a small-molecule, topical
approach to the rescue of eumelanin synthesis in a UV-
independent manner. Future studies will be needed to examine
the optimal applications of such agents in a variety of clinical
settings.
EXPERIMENTAL PROCEDURES
See the Supplemental Information for detailed methods.
Materials
SIK inhibitors were dissolved in 30% propylene glycol plus 70% ethanol. HG 9-
91-01 was purchased from Medchem Express, and all other SIK inhibitors
were synthesized by the authors.
Kinome Profiling
Kinome profiling was performed using KinomeScan ScanMAX at a compound
concentration of 1 mM. Data are reported in the Supplemental Information.
Protocols are available from DiscoverX.
Kinase Activity In Vitro Assay
The biochemical activities against SIK2 were measured with a Caliper-based
mobility shift assay (PerkinElmer).
Real-Time qPCR
The relative expression of each gene was calculated with 7500 Fast Real-
Time PCR System software, which utilizes Ct normalized to mRNA levels of
RPL11 to calculate relative expression. Results are reported relative to control
cells.
Mice
C57BL/6J Mc1r
e/e
mice were crossed with K14-SCF transgenic mice, and
C57BL/6J Tyr
c2j/c2j
were crossed with K14-SCF transgenic mice (D’Orazio
et al., 2006; Kunisada et al., 1998). Mixed-gender adult mice were used.
All animal experiments were performed in accordance with institutional
policies and Institutional Animal Care and Use Committee-approved
protocols.
Human Tissue Samples
Skin samples considered surgical waste were obtained de-identified from
healthy donors undergoing reconstructive surgery, according to institutional
regulation.
Colorimeter Measurements
Differences in darkening of the skin were measured by reflective colorimetry
(Commission Internationale de l’Eclairage [CIE] L* white-black color axis) utiliz-
ing a CR-400 Colorimeter (Minolta) calibrated to a white standard background
calibration plate, with calibration date set to Y 93.1, x 0.3133, y 0.3194, before
each set of measurements.
Statistical Analysis
Data are presented as the mean ±SEM.
Statistical significance of differences between experimental groups for
in vitro experiments of cell lines treated with varying doses of SIKi or vehicle
control were assessed by one-way ANOVA with Dunnett’s multiple compari-
sons post-test. In vitro time course experiments were assessed by
repeated-measures one-way ANOVA with Dunnett’s multiple comparisons
post-test.
Statistical significance for colorimeter readings in Figure 2B was determined
by multiple t test analysis between day 0 and day 7 for each treatment group,
with the two-stage linear step-up procedure of Benjamini, Krieger, and Yeku-
tieli to correct for false discovery rate (FDR)—desired FDR (Q) = 1%—with no
assumption of consistent SD. Statistical significance for colorimeter readings
in Figure S3B were assessed by one-way ANOVA with Dunnett’s multiple
comparisons post-test.
For the G361 melanoma cells transduced with LKB1, a one-way
ANOVA was used, with Dunnett’s multiple comparisons test to asse ss the
statistical significance of LKB1 expression and a two-t ailed paired t test
to assess the statistical significance of MITF induction with SIK-inhibitor
treatment.
Statistical significance of differences between experimental groups for
in vivo time course experiments was assessed by two-way ANOVAs with
Sidak’s multiple comparisons test.
Multiplicity-adjusted p values were reported for each comparison, and
differences of means were considered significant if p < 0.05.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
three figures, and one data file and can be found with this article online at
AUTHOR CONTRIBUTIONS
N.M., Y.L., R.M., N.S.G., and D.E.F. contributed to conception and design of
the study; N.M., Y.L., H.G.C., A.S.D., J.W., Y.S., Q.Y.W., J.A., L.V.K., A.L.H.,
and E.M.R. contributed to acquisition and analysis of data. N.M., D.E.F., and
J.W. wrote the article.
ACKNOWLEDGMENTS
The authors thank Nicholas Lowe, Xunwei Wu, Jie Wen, Yang Feng, and Vivien
Igras for technical advice and assistance. This work was supported by grants
from NIH (5P01 CA163222 and 5R01 AR043369-19), the Melanoma Research
Alliance, the Dr. Miriam and Sheldon G. Adelson Medical Research Founda-
tion, and the Canadian Institutes of Health Research (DFS-140391).
2182 Cell Reports 19, 2177–2184, June 13, 2017
N.M., R.M., N.S.G., and D.E.F. declare that parts of the work are subject
of a U.S. provisional patent application titled ‘‘Pyrimidopyrimidinones as SIK
Inhibitors.’’
Received: March 16, 2017
Revised: May 2, 2017
Accepted: May 12, 2017
Published: June 13, 2017
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