TY - JOUR
T1 - Eg5 Inhibitors Have Contrasting Effects on Microtubule Stability and Metaphase Spindle Integrity
AU - Chen, Geng Yuan
AU - Kang, You Jung
AU - Gayek, A. Sophia
AU - Youyen, Wiphu
AU - Tüzel, Erkan
AU - Ohi, Ryoma
AU - Hancock, William O.
N1 - Publisher Copyright:
© 2017 American Chemical Society.
PY - 2017/4/21
Y1 - 2017/4/21
N2 - To uncover their contrasting mechanisms, antimitotic drugs that inhibit Eg5 (kinesin-5) were analyzed in mixed-motor gliding assays of kinesin-1 and Eg5 motors in which Eg5 "braking" dominates motility. Loop-5 inhibitors (monastrol, STLC, ispinesib, and filanesib) increased gliding speeds, consistent with inducing a weak-binding state in Eg5, whereas BRD9876 slowed gliding, consistent with locking Eg5 in a rigor state. Biochemical and single-molecule assays demonstrated that BRD9876 acts as an ATP- and ADP-competitive inhibitor with 4 nM KI. Consistent with its microtubule polymerase activity, Eg5 was shown to stabilize microtubules against depolymerization. This stabilization activity was eliminated in monastrol but was enhanced by BRD9876. Finally, in metaphase-arrested RPE-1 cells, STLC promoted spindle collapse, whereas BRD9876 did not. Thus, different Eg5 inhibitors impact spindle assembly and architecture through contrasting mechanisms, and rigor inhibitors may paradoxically have the capacity to stabilize microtubule arrays in cells.
AB - To uncover their contrasting mechanisms, antimitotic drugs that inhibit Eg5 (kinesin-5) were analyzed in mixed-motor gliding assays of kinesin-1 and Eg5 motors in which Eg5 "braking" dominates motility. Loop-5 inhibitors (monastrol, STLC, ispinesib, and filanesib) increased gliding speeds, consistent with inducing a weak-binding state in Eg5, whereas BRD9876 slowed gliding, consistent with locking Eg5 in a rigor state. Biochemical and single-molecule assays demonstrated that BRD9876 acts as an ATP- and ADP-competitive inhibitor with 4 nM KI. Consistent with its microtubule polymerase activity, Eg5 was shown to stabilize microtubules against depolymerization. This stabilization activity was eliminated in monastrol but was enhanced by BRD9876. Finally, in metaphase-arrested RPE-1 cells, STLC promoted spindle collapse, whereas BRD9876 did not. Thus, different Eg5 inhibitors impact spindle assembly and architecture through contrasting mechanisms, and rigor inhibitors may paradoxically have the capacity to stabilize microtubule arrays in cells.
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U2 - 10.1021/acschembio.6b01040
DO - 10.1021/acschembio.6b01040
M3 - Article
C2 - 28165699
AN - SCOPUS:85018495823
SN - 1554-8929
VL - 12
SP - 1038
EP - 1046
JO - ACS chemical biology
JF - ACS chemical biology
IS - 4
ER -