title: STLC-resistant cell lines as tools to classify chemically divergent Eg5 targeting agents according to their mode of action and target specificity.
abstract: Determining the mechanism of action of drugs and their target specificity in cells remains a major challenge. Here we describe the use of cell lines expressing two point mutations in the allosteric inhibitor binding pocket of the mitotic kinesin Eg5 (D130A, in the loop L5 region and L214A in helix α3), which following transfection, were selected for their ability to proliferate normally in the presence of STLC, a well known Eg5 inhibitor. The cell lines were used to discriminate the mechanism of action of other chemically distinct small molecule inhibitors of Eg5 that differ in their mode of action. The STLC resistant cells were capable of continuous proliferation in the presence of ATP uncompetitive inhibitors, such as K858 and dimethylenastron, but were still sensitive to ATP competitive inhibitors that are thought to bind to a distinct site on Eg5 than the allosteric binding pocket. The STLC resistant cell lines can therefore be used as a filter to distinguish Eg5 loop L5 binding drugs from drugs binding to other pockets without prior structural information. Additionally, the cells can be used to analyze whether inhibitors of Eg5 are specific to this potential drug target or whether they have additional targets in dividing cells.
authors.affiliation: Univ. Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38027 Grenoble, France; CEA, DSV, IBS, F-38027 Grenoble, France; CNRS, IBS, F-38027 Grenoble, France.
authors.forename: Rose-Laure
authors.initials: RL
authors.lastname: Indorato
authors.forename: Salvatore
authors.initials: S
authors.lastname: DeBonis
authors.forename: Frank
authors.initials: F
authors.lastname: Kozielski
authors.forename: Isabel
authors.initials: I
authors.lastname: Garcia-Saez
authors.forename: Dimitrios A
authors.initials: DA
authors.lastname: Skoufias
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000495:Allosteric Site; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001665:Binding Sites; D045744:Cell Line, Tumor / Q000187:drug effects* / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D059566:M Phase Cell Cycle Checkpoints / Q000187:drug effects; D017354:Point Mutation; D011799:Quinazolines / Q000494:pharmacology; D013830:Thiadiazoles / Q000494:pharmacology; D013871:Thiones / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; C541942:K 858; C470243:KIF11 protein, human; D011799:Quinazolines; D013830:Thiadiazoles; D013871:Thiones; C518969:dimethylenastron; C003724:3-tritylthio-L-alanine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins; D003545:Cysteine
keywords: ATP-competitive inhibitors; ATP-uncompetitive inhibitors; Eg5 motor protein; Mitosis; Resistance to antimitotic agents; S-trityl-l-cysteine