title: cAMP-dependent activation of protein kinase A as a therapeutic target of skin hyperpigmentation by diphenylmethylene hydrazinecarbothioamide.
abstract: OBJECTIVE
  cAMP as a second messenger stimulates expression of microphthalmia-associated transcription factor (MITF) or the tyrosinase gene in UVB-induced skin pigmentation. Diphenylmethylene hydrazinecarbothioamide (QNT 3-80) inhibits α-melanocyte-stimulating hormone (α-MSH)-induced melanin production in B16 murine melanoma cells but its molecular basis remains to be defined. Here, we investigated the mechanism underlying the amelioration of skin hyperpigmentation by QNT 3-80.
  
  
  METHODS
  We used melanocyte cultures with raised levels of cAMP and UVB-irradiated dorsal skin of guinea pigs for pigmentation assays. Immunoprecipitation, kemptide phosphorylation, fluorescence analysis and docking simulation were applied to elucidate a molecular mechanism of QNT 3-80.
  
  
  RESULTS
  QNT 3-80 inhibited melanin production in melanocyte cultures with elevated levels of cAMP, including those from human foreskin. This compound also ameliorated hyperpigmentation in vivo in UVB-irradiated dorsal skin of guinea pigs. As a mechanism, QNT 3-80 directly antagonized cAMP binding to the regulatory subunit of PKA, nullified the dissociation and activation of inactive PKA holoenzyme in melanocytes and fitted into the cAMP-binding site on the crystal structure of human PKA under the most energetically favourable simulation. QNT 3-80 consequently inhibited cAMP- or UVB-induced phosphorylation (activation) of cAMP-responsive element-binding protein in vitro and in vivo, thus down-regulating expression of genes for MITF or tyrosinase in the melanogenic process.
  
  
  CONCLUSIONS
  Our data suggested that QNT 3-80 could contribute significantly to the treatment of skin disorders with hyperpigmented patches with the cAMP-binding site of PKA as its molecular target.
authors.affiliation: College of Pharmacy, Chungbuk National University, Cheongju, Korea.
authors.forename: Hyoeun
authors.initials: H
authors.lastname: Shin
authors.affiliation: College of Pharmacy, Chungbuk National University, Cheongju, Korea.
authors.forename: Seung Deok
authors.initials: SD
authors.lastname: Hong
authors.affiliation: College of Pharmacy, Chungbuk National University, Cheongju, Korea.
authors.forename: Eunmiri
authors.initials: E
authors.lastname: Roh
authors.affiliation: College of Pharmacy, Chungnam National University, Daejeon, Korea.
authors.forename: Sang-Hun
authors.initials: SH
authors.lastname: Jung
authors.affiliation: College of Pharmacy, Chonnam National University, Gwangju, Korea.
authors.forename: Won-Jea
authors.initials: WJ
authors.lastname: Cho
authors.affiliation: College of Pharmacy, Chungbuk National University, Cheongju, Korea.
authors.forename: Sun Hong
authors.initials: SH
authors.lastname: Park
authors.affiliation: College of Pharmacy, Chungbuk National University, Cheongju, Korea.
authors.forename: Da Young
authors.initials: DY
authors.lastname: Yoon
authors.affiliation: College of Pharmacy, Chungbuk National University, Cheongju, Korea.
authors.forename: Seon Mi
authors.initials: SM
authors.lastname: Ko
authors.affiliation: College of Pharmacy, Chungbuk National University, Cheongju, Korea.
authors.forename: Bang Yeon
authors.initials: BY
authors.lastname: Hwang
authors.affiliation: College of Pharmacy, Chungbuk National University, Cheongju, Korea.
authors.forename: Jin Tae
authors.initials: JT
authors.lastname: Hong
authors.affiliation: College of Natural Sciences, Chungbuk National University, Cheongju, Korea.
authors.forename: Tae-Young
authors.initials: TY
authors.lastname: Heo
authors.affiliation: College of Pharmacy, Chungbuk National University, Cheongju, Korea.
authors.forename: Sang-Bae
authors.initials: SB
authors.lastname: Han
authors.affiliation: College of Pharmacy, Chungbuk National University, Cheongju, Korea.
authors.forename: Youngsoo
authors.initials: Y
authors.lastname: Kim
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D015536:Down-Regulation; D052816:Foreskin / Q000166:cytology; D006168:Guinea Pigs; D006801:Humans; D017495:Hyperpigmentation / Q000188:drug therapy; D008297:Male; D008543:Melanins / Q000378:metabolism; D008544:Melanocytes / Q000187:drug effects / Q000378:metabolism; D051739:Microphthalmia-Associated Transcription Factor / Q000235:genetics; D014442:Monophenol Monooxygenase / Q000235:genetics; D012867:Skin / Q000528:radiation effects; D012880:Skin Pigmentation / Q000187:drug effects / Q000502:physiology*; D013882:Thiosemicarbazones / Q000494:pharmacology*; D014466:Ultraviolet Rays
chemical_list: D008543:Melanins; D051739:Microphthalmia-Associated Transcription Factor; D013882:Thiosemicarbazones; C000606309:diphenylmethylene hydrazinecarbothioamide; D000242:Cyclic AMP; D014442:Monophenol Monooxygenase; D017868:Cyclic AMP-Dependent Protein Kinases