Melanocortins and the melanocortin 1 receptor, moving translationally towards melanoma prevention.

Beginning in the last decade of the twentieth century, the fields of pigment cell research and melanoma have witnessed major breakthroughs in the understanding of the role of melanocortins in human pigmentation and the DNA damage response of human melanocytes to solar ultraviolet radiation (UV). This began with the cloning of the melanocortin 1 receptor (MC1R) gene from human melanocytes and the demonstration that the encoded receptor is functional. Subsequently, population studies found that the MC1R gene is highly polymorphic, and that some of its variants are associated with red hair phenotype, fair skin and poor tanning ability. Using human melanocytes cultured from donors with different MC1R genotypes revealed that the alleles associated with red hair color encode for a non-functional receptor. Epidemiological studies linked the MC1R red hair color variants to increased melanoma risk. Investigating the impact of different MC1R variants on the response of human melanocytes to UV led to the important discovery that the MC1R signaling activates antioxidant, DNA repair and survival pathways, in addition to stimulation of eumelanin synthesis. These effects of MC1R were absent in melanocytes expressing 2 MC1R red hair color variants that result in loss of function of the receptor. The importance of the MC1R in reducing UV-induced genotoxicity in melanocytes led us to design small peptide analogs of the physiological MC1R agonist α-melanocortin (α-melanocyte stimulating hormone; α-MSH) for the goal of utilizing them for melanoma chemoprevention.

Canonical Bcl-2 motifs of the Na+/K+ pump revealed by the BH3 mimetic chelerythrine: early signal transducers of apoptosis?

BACKGROUND/AIMS: Chelerythrine [CET], a protein kinase C [PKC] inhibitor, is a prop-apoptotic BH3-mimetic binding to BH1-like motifs of Bcl-2 proteins. CET action was examined on PKC phosphorylation-dependent membrane transporters (Na+/K+ pump/ATPase [NKP, NKA], Na+-K+-2Cl+ [NKCC] and K+-Cl- [KCC] cotransporters, and channel-supported K+ loss) in human lens epithelial cells [LECs]. METHODS: K+ loss and K+ uptake, using Rb+ as congener, were measured by atomic absorption/emission spectrophotometry with NKP and NKCC inhibitors, and Cl- replacement by NO3ˉ to determine KCC. 3H-Ouabain binding was performed on a pig renal NKA in the presence and absence of CET. Bcl-2 protein and NKA sequences were aligned and motifs identified and mapped using PROSITE in conjunction with BLAST alignments and analysis of conservation and structural similarity based on prediction of secondary and crystal structures. RESULTS: CET inhibited NKP and NKCC by >90% (IC50 values ~35 and ~15 µM, respectively) without significant KCC activity change, and stimulated K+ loss by ~35% at 10-30 µM. Neither ATP levels nor phosphorylation of the NKA α1 subunit changed. 3H-ouabain was displaced from pig renal NKA only at 100 fold higher CET concentrations than the ligand. Sequence alignments of NKA with BH1- and BH3-like motifs containing pro-survival Bcl-2 and BclXl proteins showed more than one BH1-like motif within NKA for interaction with CET or with BH3 motifs. One NKA BH1-like motif (ARAAEILARDGPN) was also found in all P-type ATPases. Also, NKA possessed a second motif similar to that near the BH3 region of Bcl-2. CONCLUSION: Findings support the hypothesis that CET inhibits NKP by binding to BH1-like motifs and disrupting the α1 subunit catalytic activity through conformational changes. By interacting with Bcl-2 proteins through their complementary BH1- or BH3-like-motifs, NKP proteins may be sensors of normal and pathological cell functions, becoming important yet unrecognized signal transducers in the initial phases of apoptosis. CET action on NKCC1 and K+ channels may involve PKC-regulated mechanisms; however, limited sequence homologies to BH1-like motifs cannot exclude direct effects.

Development of hMC1R Selective Small Agonists for Sunless Tanning and Prevention of Genotoxicity of UV in Melanocytes.

Activation of the human melanocortin 1 receptor (hMC1R) expressed on melanocytes by α-melanocortin plays a central role in regulating human pigmentation and reducing the genotoxicity of UV by activating DNA repair and antioxidant defenses. For the development of a hMC1R-targeted photoprotection strategy, we designed tetra- and tripeptide agonists with modifications that provide the necessary lipophilicity and hMC1R selectivity to be effective drugs. These peptides proved to be superior to most of the existing analogs of the physiological tridecapeptide α-melanocortin because of their small size and high hMC1R selectivity. Testing on primary cultures of human melanocytes showed that these peptides are highly potent with prolonged stimulation of melanogenesis, enhanced repair of UV-induced DNA photoproducts, and reduced apoptosis. One of the tripeptides, designated as LK-514 (5), with a molecular weight of 660 Da, has unprecedented (>100,000) hMC1R selectivity when compared with the other melanocortin receptors hMC3R, hMC4R, and hMC5R, and increases pigmentation (sunless tanning) in a cultured, three-dimensional skin model. These new analogs should be efficacious in preventing skin cancer, including melanoma, and treatment of skin disorders, such as vitiligo and polymorphic light eruptions.

Exploring the PI3K alpha and gamma binding sites with 2,6-disubstituted isonicotinic derivatives.

A homology model of the p110alpha catalytic subunit of PI3Kalpha was generated from the p110gamma crystal structure. Using this model, an isonicotinic scaffold was designed for chemically exploring the PI3Kalpha and gamma binding sites. A focused library of derivatives was synthesized and tested. The morpholine acids 5a and 5b proved to be the most potent analogs.

Semi-rigid tripeptide agonists of melanocortin receptors.

A series of 30 RCO-HfR-NH(2) derivatives show preference for the mouse MC1R vs MC3-5Rs. trans-4-HOC(6)H(4)CH=CHCO-HfR-NH(2) (13) [EC(50) (nM): MC1R 83, MC3R 20500, MC4R 18130 and MC5R 935; ratio 1:246:217:11] is 11 times more potent than the lead compound LK-394 Ph(CH(2))(3)CO-HfR-NH(2) (2) and only 11 times less potent than the native tridecapeptide alpha-MSH at mMC1R. Differences in conformations of 2 and 13 are discussed.

Melanoma prevention strategy based on using tetrapeptide alpha-MSH analogs that protect human melanocytes from UV-induced DNA damage and cytotoxicity.

Melanoma is the deadliest form of skin cancer, with no cure for advanced disease. We propose a strategy for melanoma prevention based on using analogs of alpha-melanocyte stimulating hormone (alpha-MSH) that function as melanocortin 1 receptor (MC1R) agonists. Treatment of human melanocytes with alpha-MSH results in stimulation of eumelanin synthesis, reduction of apoptosis that is attributable to reduced hydrogen peroxide generation and enhanced repair of DNA photoproducts. These effects should contribute to genomic stability of human melanocytes, thus preventing their malignant transformation to melanoma. Based on these findings, we synthesized and tested the effects of 3 tetrapeptide alpha-MSH analogs, Ac-His-D-Phe-Arg-Trp-NH2, n-Pentadecanoyl- and 4-Phenylbutyryl-His-D-Phe-Arg-Trp-NH2, on cultured human melanocytes. The latter two analogs were more potent than the former, or alpha-MSH, in stimulating the activity of tyrosinase, thus melanogenesis, reducing apoptosis and release of hydrogen peroxide and enhancing repair of DNA photoproducts in melanocytes exposed to UV radiation (UVR). The above analogs are MC1R agonists, as their effects were abrogated by an analog of agouti signaling protein, the physiological MC1R antagonist, and were absent in melanocytes expressing loss-of-function MC1R. Analogs, such as 4-Phenylbutyryl-His-D-Phe-Arg-Trp-NH2 with prolonged and reversible effects, can potentially be developed into topical agents to prevent skin photocarcinogenesis, particularly melanoma.

A one-pot synthesis of beta-C-glucopyranosides from exo-glucal, p-tolylsulfenyl chloride, an alpha-methoxyalkene, and an external nucleophile.

[formula: see text] beta-C-Glycosides were synthesized in one-pot experiments using the following sequence of four reactions: (i) addition of p-TolSCl to an alpha-methoxyalkene, (ii) generation of the episulfonium ion from a beta-(arylsulfanyl)alkyl chloride, (iii) reaction of the episulfonium intermediate with benzylated exo-D-glucal to form a cyclic five-membered sulfonium salt, and (iv) quenching of the sulfonium salt with the external nucleophile: H2O, CH3OH, or NaCNBH3.

One-pot synthesis of C-glycosylic compounds (C-glycosides) from D-glucal, p-tolylsulfenyl chloride and aromatic/heteroaromatic compounds in the presence of Lewis acids.

In the presence of Zn(CN)(2), benzylated 2-thio-2-S-(p-tolyl)pyranosyl chlorides (2) generated in situ from tri-O-benzyl-D-glucal and p-TolSCl, smoothly react with thiophene, 2-methylthiophene, furan, 2-methylfuran, and N-methylpyrrole to give heteroaryl 2-thio-2-S-(p-tolyl)-C-beta-D-glucopyranosylic compounds (C-glycosides) in good yields. Upon treatment with SnCl(4), the reaction of chlorides 2 with thiophene or 1,4-dimethoxybenzene provides the corresponding benzylated C-beta-D-glucofuranosylic derivatives. Under the same conditions, the use of 2-methylthiophene, furan, 2-methylfuran, or N-methylpyrrole yields (2S,3R,4R,5S)-1,3,4-tribenzyloxy-6,6-diheteroaryl-5-(p-tolylthio)-2-hexanoles. Treatment of 2 and mesitylene with AgBF(4) yielded 1,6-anhydro-3,4-di-O-benzyl-2-thio-2-S-(p-tolyl)-beta-D-glucose.

alpha-MSH tripeptide analogs activate the melanocortin 1 receptor and reduce UV-induced DNA damage in human melanocytes.

One skin cancer prevention strategy that we are developing is based on synthesizing and testing melanocortin analogs that reduce and repair DNA damage resulting from exposure to solar ultraviolet (UV) radiation, in addition to stimulating pigmentation. Previously, we reported the effects of tetrapeptide analogs of alpha-melanocortin (alpha-MSH) that were more potent and stable than the physiological alpha-MSH, and mimicked its photoprotective effects against UV-induced DNA damage in human melanocytes. Here, we report on a panel of tripeptide analogs consisting of a modified alpha-MSH core His(6)-d-Phe(7)-Arg(8), which contained different N-capping groups, C-terminal modifications, or arginine mimics. The most potent tripeptides in activating cAMP formation and tyrosinase of human melanocytes were three analogs with C-terminal modifications. The most effective C-terminal tripeptide mimicked alpha-MSH in reducing hydrogen peroxide generation and enhancing nucleotide excision repair following UV irradiation. The effects of these three analogs required functional MC1R, as they were absent in human melanocytes that expressed non-functional receptor. These results demonstrate activation of the MC1R by tripeptide melanocortin analogs. Designing small analogs for topical delivery should prove practical and efficacious for skin cancer prevention.