In vitro activity: BTB-1 is a novel, potent and the first small molecule inhibitor of the mitotic motor protein Kif18A. It is selective within the kinesin subgroup of Kif4, Eg5, MKLP-1, MKLP-2, MPP1, CENP-E and MCAK. Kinesin motors play diverse roles in mitosis and are targets for antimitotic drugs. Since Kif18A is frequently overexpressed in solid tumors, compounds like BTB-1 are not only of great interest for basic research but also have the potential to open up new strategies for the treatment of human diseases. During in vitro motility assays Kif18A’s MT gliding ability was blocked by the addition of BTB-1 in a reversible manner. Strikingly, BTB-1 binding site is close to that of well-characterized Kif11 inhibitors that block tight microtubule binding, whereas BTB-1 traps Kif18A on the microtubule.

Kinase Assay: BTB-1 is prepared in DMSO. The activity of His-Kif18Amotor at increasing concentrations of ATP is monitored in the presence of 3 μM Mts and increasing concentrations of BTB-1 (0.21 μM, 0.42 μM, 0.85 μM, 1.7 μM) or DMSO as control.

Cell Assay: BTB-1 blocks the motility of Kif18A in a reversible manner. BTB-1 inhibits Kif18A in an adenosine triphosphate (ATP)-competitive but microtubule-uncompetitive manner and slows down the progression of cells through mitosis. 100 μM BTB-1 does not significantly inhibit any of the other tested mitotic kinesins. BTB-1 competes with ATP for Kif18A binding only when the motor-protein is associated with its pseudosubstrate microtubules. HeLa cells treated with BTB-1 accumulate in mitosis in a dose-dependent manner. BTB-1 shows cell toxicity with an EC50 values of 35.8 μM. HeLa cells treated with 50 μM BTB-1 reveals severe defects in spindle morphology and chromosome alignment. Treatment with high concentrations of BTB-1 does not result in elongated spindles.