STLC-resistant cell lines as tools to classify chemically divergent Eg5 targeting agents according to their mode of action and target specificity
Graphical abstract
Eg5-D130A or Eg5-L214A U2OS expressing cells are capable of colony formation in the presence of ATP-uncompetitive Eg5 inhibitors whereas they remain sensitive to ATP competitive inhibitors.
Introduction
Microtubule targeting agents, such as taxol and vinblastine and their analogs, are currently used successfully in the clinic as chemotherapeutic agents targeting certain cancer tumor malignancies [1]. The underlying working hypothesis, is that interfering with microtubule dynamics, either by stabilizing or depolymerizing spindle microtubules, leads to mitotic arrest followed by cell death [2]. Currently though, antimitotic targeted therapy is under challenge [3] due to the lower proliferation rates of cells observed in tumors compared to cancer cell proliferation in tissue culture, an observation referred to as the proliferation rate paradox [4]. Nonetheless, there is still an interest in identifying new mitotic targets for the purpose of developing more specific mitotic inhibitors. Among the recently heavily pursued mitotic targets is the mitotic kinesin motor protein Eg5 [5].
Eg5 is a member of the kinesin superfamily of proteins all of which share structural similarities in the motor domain [6]. Eg5 is a homotetramer, formed by a staggered antiparallel coiled-coil of two homodimers [7], [8]. This bipolar nature of the Eg5 tetramer allows the crosslinking of antiparallel spindle microtubules. The microtubule plus end motor activity of Eg5 is necessary for the separation of the duplicated centrosomes during prophase and the establishment of a bipolar spindle [9]. Depending on the species, Eg5 activity is also needed for the maintenance and for regulating the length of the metaphase spindle [10], [11] and in anaphase spindle elongation [12]. Cells depleted of Eg5 fail to build a bipolar spindle and instead form a monoastral spindle characterized by an extensive array of microtubules nucleated from the two unseparated centrosomes surrounded by chromosomes [6]. The monoastral spindle is also the resulting phenotype of cells exposed to small molecule inhibitors such as monastrol, STLC and others [13], [14].
Since the discovery of monastrol [13], a plethora of Eg5 inhibitors have been characterized and used for chemical genetic purposes [15], [16]. In addition, a number of Eg5 inhibitors entered clinical trials with varying success [17], [18], [19]. Monastrol and its analogs as well as other chemically distinct small molecules such as STLC, are allosteric inhibitors known to bind to a unique pocket in the Eg5 motor domain formed by the secondary structure elements helix α2/loop L5/α3 [20], [21]. The mechanism of action of the inhibitors that bind to the allosteric loop L5 site has been shown to be ATP uncompetitive [22], [23]. Loop L5 undergoes a conformational change that traps the Eg5 targeting small molecules inside the pocket. The presence of the inhibitor in the L5 pocket eventually renders the motor domain in an “ATP-like” conformation (although ADP is bound in the catalytic side) inhibiting the mechanochemical cycle of the motor by slowing down ADP release [24]. Furthermore, a second class of Eg5 inhibitors that compete with ATP binding at or near the nucleotide binding site has been identified [25], [26]. Interestingly, some of the ATP competitive Eg5 inhibitors are shown to bind in a different allosteric site other than the STLC binding site [26].
Earlier structure activity studies have shown that certain rationally designed mutations in the loop L5 region resulted in Eg5 that was resistant to monastrol and STLC [27], [28]. The in vitro ATPase activity of the mutants was not significantly altered in the presence of the inhibitors [29], [30] and when the mutants were expressed after transient transfections the expressing cells were resistant to monastrol and to STLC [31]. To date, resistance to Eg5 inhibitors has been demonstrated to arise spontaneously only in tissue culture [25], [32]. The limited clinical data do not allow the conclusion whether or not resistance to Eg5 inhibitors through mutations is, or might be, a major issue. However, the comparison of sensitive and resistant cell lines with the same starting genetic background may be a good strategy to study mechanisms of drug action and to screen for inhibitors that bind novel binding sites on the same protein target.
In the present study we have selected clones of U2OS cells expressing either D130A or L214A Eg5 mutants using STLC as the selection agent. The clones were capable of cycling normally in the presence of STLC. We then tested the STLC resistant clones for their ability to grow in the presence of a variety of ATP uncompetitive and ATP competitive inhibitors. STLC resistant clones were capable of dividing normally in the presence of all ATP uncompetitive inhibitors tested. However, the same cell lines remained sensitive to ATP competitive inhibitors by arresting in mitosis. The results show that the STLC resistant clones may be used as a filter – without prior structural information – to distinguish loop L5 binding drugs from those that bind to other pockets on Eg5. Another very useful application is to analyze whether or not small molecule inhibitors of Eg5 are specific to Eg5 or may also target other proteins in dividing cells.
Section snippets
Selection of U2OS cells resistant to STLC
U2OS cells were grown in D-MEM (Gibco BRL; Paisley, UK), supplemented with 10% fetal calf serum (Hyclone) in 60 mm petri dishes up to 50% confluence before being transfected with either wild-type, D130A- or L214A-Eg5-pRcCMV plasmids [31] using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacture's protocol. Twenty four hours following transfection cells were exposed to 10 μM STLC (Novabiochem, Merck KGaA, Darmstadt, Germany) for four weeks; the STLC containing medium was
Characterization of STLC resistant clones expressing either Eg5-D130A or Eg5-L214A mutants
Based on the fact that exposure of cells to STLC results in cell death following mitotic failure [36], we carried out the selection of transfected U2OS cells, with wild type or point mutants of human Eg5 in pRcCMV plasmids, using STLC as the selection pressure agent instead of the standard antibiotic G418 (Geneticin). Two Eg5 point mutations were selected, D130A and L214A, in transient transfection experiments described previously [31], and based on their ability to efficiently confer
Discussion
We, and others have previously shown that certain mutations in the allosteric inhibitor binding pocket of Eg5 (helix α2/loop L5 and helix α3) can confer resistance to STLC, monastrol or other inhibitors [29], [30], [31], [38], [39]. Additionally, certain of the Eg5 mutants, when expressed in cells, they also conferred resistance to STLC and monastrol suggesting also that the inhibitors were specific to Eg5 and did not have additional mitotic targets [31]. In the present study we took advantage
Acknowledgements
We thank Françoise Lacroix and Jean-Philippe Kleman (Institut de Biologie Structurale, Grenoble) for the support and access to the Cell Imaging Platform. This work was supported by La Ligue Contre Le Cancer Comité du Rhône to DAS.
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