title: Tubulin is a molecular target of the Wnt-activating chemical probe.
abstract: In drug discovery research, cell-based phenotypic screening is an essential method for obtaining potential drug candidates. Revealing the mechanism of action is a key step on the path to drug discovery. However, elucidating the target molecules of hit compounds from phenotypic screening campaigns remains a difficult and troublesome process. Simple and efficient methods for identifying the target molecules are essential.
  
  
  
  2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) was identified as a senescence inducer from a phenotypic screening campaign. The compound is widely used as a Wnt agonist, although its target molecules remain to be clarified. To identify its target proteins, we compared a series of cellular assay results for the compound with our pathway profiling database. The database comprises the activities of compounds from simple assays of cellular reporter genes and cellular proliferations. In this database, compounds were classified on the basis of statistical analysis of their activities, which corresponded to a mechanism of action by the representative compounds. In addition, the mechanisms of action of the compounds of interest could be predicted using the database. Based on our database analysis, the compound was anticipated to be a tubulin disruptor, which was subsequently confirmed by its inhibitory activity of tubulin polymerization.
  
  
  
  These results demonstrate that tubulin is identified for the first time as a target molecule of the Wnt-activating small molecule and that this might have misled the conclusions of some previous studies. Moreover, the present study also emphasizes that our pathway profiling database is a simple and potent tool for revealing the mechanisms of action of hit compounds obtained from phenotypic screenings and off targets of chemical probes.
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraokahigashi, Fujisawa, Kanagawa, Japan.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Fukuda
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraokahigashi, Fujisawa, Kanagawa, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Sano
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraokahigashi, Fujisawa, Kanagawa, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Kazetani
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraokahigashi, Fujisawa, Kanagawa, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraokahigashi, Fujisawa, Kanagawa, Japan. hidehisa.iwata@takeda.com.
authors.forename: Hidehisa
authors.initials: H
authors.lastname: Iwata
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraokahigashi, Fujisawa, Kanagawa, Japan. junji.matsui@takeda.com.
authors.forename: Junji
authors.initials: J
authors.lastname: Matsui
mesh_terms: D052117:Benzodioxoles / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D016922:Cellular Senescence / Q000187:drug effects; D016000:Cluster Analysis; D016208:Databases, Factual; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D006801:Humans; D053858:Metabolic Networks and Pathways / Q000187:drug effects; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D011743:Pyrimidines / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism; D050257:Tubulin Modulators / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D051153:Wnt Proteins / Q000819:agonists* / Q000378:metabolism
chemical_list: C581356:2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine; D052117:Benzodioxoles; D011743:Pyrimidines; D014404:Tubulin; D050257:Tubulin Modulators; D051153:Wnt Proteins