title: Discovery of a novel neuroprotective compound, AS1219164, by high-throughput chemical screening of a newly identified apoptotic gene marker.
abstract: We have reported that tacrolimus (FK506), an immunosuppressive drug, and diclofenac, a non-steroidal anti-inflammatory drug, possess different modes of neuroprotective action. FK506 suppresses only thapsigargin-induced apoptosis in neuroblastoma SH-SY5Y cells while diclofenac reverses tunicamycin-induced as well as thapsigargin-induced apoptosis. The aim of this study is to discover novel compounds that exert neuroprotective properties by using the transcriptional response of a newly identified gene, which was regulated by both FK506 and diclofenac, as a surrogate screening marker in high-throughput chemical screening and characterize the compounds in comparison with FK506 and diclofenac. Using a microarray with 4504 human cDNAs and quantitative RT-PCR, two genes as apoptotic markers, transmembrane protein 100 (TMEM100) and limb-bud and heart (LBH), were identified because the thapsigargin-induced elevations in their mRNA levels were reversed by both FK506 and diclofenac. A luciferase reporter assay with a TMEM100 promoter region was applied to high-throughput chemical screening. AS1219164, {3-[(E)-2-{5-[(E)-2-pyridin-4-ylvinyl]pyridin-3-yl} vinyl]aniline}, suppressed thapsigargin-induced transactivation of the TMEM100 gene and reversed thapsigargin-induced increases in TMEM100 and LBH mRNA levels in SH-SY5Y cells, similar to the effects of FK506 and diclofenac. Furthermore, AS1219164 protected against SH-SY5Y cell death induced by four apoptotic agents including thapsigargin, similar to diclofenac, but was more potent than diclofenac, while FK506 only showed protective effects against thapsigargin-induced cell death. In conclusion, a novel neuroprotecitve compound, AS1219164, was discovered by high-throughput chemical screening using a reporter assay with the TMEM100 gene promoter regulated by both FK506 and diclofenac. Reporter assay using the promoter region of a gene under pharmacological and physiological transcriptional regulation would be well suit for use in high-throughput chemical screening.
authors.affiliation: Pharmacology Research Laboratories, Astellas Pharma Inc., 5-2-3, Tokodai Tsukuba, Ibaraki 300-2698, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Yamazaki
authors.forename: Masakazu
authors.initials: M
authors.lastname: Muramoto
authors.forename: Osamu
authors.initials: O
authors.lastname: Okitsu
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Morikawa
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kita
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D015415:Biomarkers; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004008:Diclofenac / Q000494:pharmacology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter / Q000187:drug effects / Q000235:genetics; D057166:High-Throughput Screening Assays; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D018696:Neuroprotective Agents / Q000494:pharmacology*; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D011725:Pyridines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D016559:Tacrolimus / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics*; D014157:Transcription Factors
chemical_list: C569641:3-(2-(5-(2-pyridin-4-ylvinyl)pyridin-3-yl)vinyl)aniline; D000814:Aniline Compounds; D015415:Biomarkers; D007166:Immunosuppressive Agents; C531176:LBH protein, human; D018696:Neuroprotective Agents; D011725:Pyridines; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; D004008:Diclofenac; D019284:Thapsigargin; D008156:Luciferases; D053148:Caspase 3; D016559:Tacrolimus