title: Genetic approach to evaluate specificity of small molecule drug candidates inhibiting PLK1 using zebrafish.
abstract: During the preclinical drug discovery process it remains a challenge to enable early elimination of candidate molecules that may have non-specific, off-target activities. Here, we use whole zebrafish embryo assays coupled with genetic analysis to address this issue. PLK1 (Polo-like kinase 1) is one of the key regulators that control mitotic entry, spindle assembly, chromosome segregation, and cytokinesis in the cell cycle. Since plk1 expression is abnormally up-regulated in several tumors, it is regarded as a good target for cancer therapy. A number of small-molecule inhibitors targeting PLK1 have been developed as reagents and anticancer drug candidates. It will be interesting to determine if these inhibitors indeed specifically target PLK1 in vivo. Bioinformatics analysis revealed that the zebrafish and human genomes share high homology across all PLK family members. In particular, PLK1 has a nearly identical 3-D structure between zebrafish and human. We selected three published PLK1 inhibitors, LFM-A13, ON01910, and thiazole-carboxamide 10A in our assay. When added at 2-cell stage, all of these inhibitors prevented embryos from dividing and caused cells to fuse into one large cell. When added at the later stage during zygotic mRNA transcription program initiation, embryos survived for 3 days but showed different phenotypes for each compound. Embryos treated with LFM-A13 appeared relatively normal. Embryos treated with ON01910 failed to properly develop trunk and tail regions while the head structure was unaffected. Embryos treated with thiazole-carboxamide 10A had a shorter body axis and deformed head structure. To determine which inhibitor is more selectively targeting PLK1, we inhibited PLK1 activity using anti-sense morpholino. Comparative analysis indicated that thiazole-carboxamide 10A could faithfully phenocopy zebrafish embryos genetically deficient of plk1. These findings demonstrate that these three PLK1 inhibitors, although well established by in vitro studies, have different off-target activities in vivo, and that thiazole-carboxamide 10A appears most specific to PLK1. Our studies suggest that zebrafish should be generally useful as an efficient in vivo model to evaluate specificity of small molecules designed to regulate any conserved target proteins through comparative analysis of genetic phenotypes.
authors.affiliation: Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Shenzhen Graduate School of Peking University, Shenzhen University Town, Shenzhen, China.
authors.forename: Hanbing
authors.initials: H
authors.lastname: Zhong
authors.forename: Shengchang
authors.initials: S
authors.lastname: Xin
authors.forename: Yanqiu
authors.initials: Y
authors.lastname: Zhao
authors.forename: Jing
authors.initials: J
authors.lastname: Lu
authors.forename: Song
authors.initials: S
authors.lastname: Li
authors.forename: Jianxian
authors.initials: J
authors.lastname: Gong
authors.forename: Zhen
authors.initials: Z
authors.lastname: Yang
authors.forename: Shuo
authors.initials: S
authors.lastname: Lin
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D020650:Combinatorial Chemistry Techniques; D017124:Conserved Sequence; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004625:Embryo, Nonmammalian; D047108:Embryonic Development / Q000187:drug effects; D005821:Genetic Techniques; D008954:Models, Biological; D010802:Phylogeny; D047428:Protein Kinase Inhibitors / Q000032:analysis / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D054852:Small Molecule Libraries / Q000032:analysis; D013379:Substrate Specificity; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D000097869:Polo-Like Kinase 1
chemical_list: D018797:Cell Cycle Proteins; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D054852:Small Molecule Libraries; D000097869:Polo-Like Kinase 1