1. Identification of Novel Pigmentation Modulators by Chemical Genetic Screening 
Li Ni-Komatsu, Seth J. Orlow 
Journal of Investigative Dermatology 
Volume 127, Issue 7, Pages (July 2007)
DOI: /sj.jid
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Novel tagged-triazine libraries and the screening procedure. (a) Structures of the triazine libraries with a TG, MP, AP, or PP linker. The four linkers are each shown in a different color. R1 and R2 groups vary among different compounds. (b) Flowchart of the screening procedure. Screening was performed with cultured melanocytes (in 24-well plates) or zebrafish eggs (in 96-well plates) followed by melanin assay (melanocytes) or microscopic imaging (zebrafish). Using the compound bound to agarose beads as an affinity matrix, the cellular binding target of each active compound was identified.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Identification of a pigmentation enhancer that binds prohibitin in a cell-based screening. (a) Chemical structure and EC50 of pigmentation enhancer melanogenin (TGV28). (b) Melanogenin induces melanin synthesis in a dose-dependent manner in immortalized murine melanocytes (melan-a cells). Melanin content is normalized to the total amount of protein in the cell lysates. (c) Melanogenin binds prohibitin specifically. Silver-stained 5–15% gradient SDS–PAGE gel illustrates melanogenin-specific protein binding. A is the unconjugated agarose bead matrix, TGV28-A is the melanogenin-conjugated affinity matrix, and negative-A is the negative control compound conjugated affinity matrix. Arrow indicates the protein band that specifically binds to melanogenin-conjugated matrix. (d) Antiprohibitin Western blot from melanocyte lysate incubated with melanogenin-conjugated agarose beads (TGV28) or agarose beads alone (A). Melanogenin-prohibitin binding was abolished on preincubation of cell lysate with free melanogenin but not with inactive control compound (negative). (e) Prohibitin gene silencing effectively attenuates the cellular response to melanogenin. Melanin assay of B16-F10 melanoma cells transfected with two distinct prohibitin siRNA sequences (Phb1 and Phb2), negative control siRNA, or irrelevant Lamin A/C siRNA followed by no treatment (control), 5μm melanogenin, or 100μm isobutylmethylxanthine treatment.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Identification of novel Tyr inhibitors by screening the TG-tagged triazine library in immortalized murine melanocytes. (a) Chemical structures and IC50 of four novel pigmentation inhibitors, TGH11, TGD10, TGD39, TGJ29, and two known pigmentation inhibitors, hydroquinone and phenylthiourea. (b) Effects of novel triazine pigmentation inhibitors on melanin synthesis in melan-a cells. The final concentration of each compound was 10μm. Data are presented as the percentage of control. (c) Identification of Tyr as the cellular target of the novel pigmentation inhibitors by affinity chromatography. Eluted proteins were separated by 7.5% SDS–PAGE and subjected to Western blot analysis. The membrane was probed with αPep7, a rabbit antisera against mouse Tyr (Jimenez et al., 1991). D10, D39, and J29 are each individual compound-conjugated affinity matrix; lysate is the melan-a cell lysates.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Identification of compounds that bind mitochondrial F1F0-ATPase for correction of OCA2 albinism by cell-based screening. (a) Chemical structures and EC50 of compounds that correct albinism. (b) Immunomicroscopic analysis of the effect of compounds that correct albinism on the distribution of Tyr and Tyr-related protein 1 in OCA2 murine melanocytes (melan-p cells).
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Identification of a pigmentation enhancer that binds mitochondrial F1F0-ATPase by screening the PP-tagged triazine library in zebrafish. (a) Chemical structures and EC50 of a pigmentation enhancer (PPA) identified by screening in zebrafish. (b) Effects of PPA on pigmentation in zebrafish embryo. Treatment with PPA induced a dose-dependent increase in pigmentation throughout the embryo. Increased pigmentation was most notable in the body of the fish embryo just caudal to the developing head (see boxes). (c) PPA increases melanin synthesis in melan-a melanocytes, albino melan-p melanocytes, and B16-F10 melanoma cells.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Cellular binding targets and proposed roles of the newly identified pigmentation modulators in the regulation of Tyr transcription, translation, processing, and trafficking in melanocytes. Melanogenin (TGV28) binds to prohibitin (Phb), a scaffold protein that interacts with various transcriptional factors (TF). Treatment with melanogenin may result in the release of transcriptional factors bound to prohibitin, allowing them to traffic to the nucleus, thus altering the level of Tyr gene transcription. TGH11, TGD10, TGD39, and TGJ29 are a novel class of potent Tyr inhibitors that most probably compete with l-3, 4-dihydroxyphenylalanine for the dihydroxyphenylalanine-specific site of Tyr. PPA, MPC11, MPD11, APC25, APC32, PPA01, and PPJ01 induce melanin synthesis by inhibiting mitochondrial F1F0-ATPase-mediated transport of H+ ions, resulting in the alkalinization of the cytosol.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions