ABSTRACT
Using a chemical genetics screen, we have identified ent-15-oxokaurenoic acid (EKA) as a chemical that causes prolonged mitotic arrest at a stage resembling prometaphase. EKA inhibits the association of the mitotic motor protein centromeric protein E with kinetochores and inhibits chromosome movement. Unlike most antimitotic agents, EKA does not inhibit the polymerization or depolymerization of tubulin. To identify EKA-interacting proteins, we used a cell-permeable biotinylated form that retains biological activity to isolate binding proteins from living cells. Mass spectrometric analysis identified six EKA-binding proteins, including Ran-binding protein 2, a kinetochore protein whose depletion by small interfering RNA causes a similar mitotic arrest phenotype.
Subject(s)
Chromosomes/ultrastructure , Diterpenes, Kaurane/chemistry , Diterpenes, Kaurane/pharmacology , Kinetochores/metabolism , Mitosis , Molecular Chaperones/metabolism , Nuclear Pore Complex Proteins/metabolism , Biotinylation , Cell Line, Tumor , Chemistry/methods , HeLa Cells , Humans , Imaging, Three-Dimensional , Mass Spectrometry , Polymers/chemistry , Protein Binding , Spindle Apparatus , Tubulin/chemistryABSTRACT
A phenotypic cell-based assay for inhibitors of the G (2) DNA damage checkpoint was used to screen plant extracts from the US National Cancer Institute Natural Products Repository. It revealed activity in a methanol extract from the common ragweed Ambrosia artemisiifolia. Assay-guided fractionation led to the identification of the sesquiterpene lactones psilostachyins A and C as novel checkpoint inhibitors. Elimination of their alpha,beta-unsaturated carbonyl group caused a loss of activity, suggesting that the compounds can bind covalently to target proteins through Michael addition. Psilostachyins A and C also blocked cells in mitosis and caused the formation of aberrant microtubule spindles. However, the compounds did not interfere with microtubule polymerization in vitro. The related sesquiterpene lactones psilostachyin B, paulitin and isopaulitin were also isolated from the same extract but showed no checkpoint inhibition. The identification of the target(s) of psilostachyins A and C may provide further insight into the signalling pathways involved in cell cycle arrest and mitotic progression.
