KIF15 nanomechanics and kinesin inhibitors, with implications for cancer chemotherapeutics

Abstract

Eg5, a mitotic kinesin, has been a target for anticancer drug development. Clinical trials of small-molecule inhibitors of Eg5 have been stymied by the development of resistance, attributable to mitotic rescue by a different endogenous kinesin, KIF15. Compared with Eg5, relatively little is known about the properties of the KIF15 motor. Here, we employed single-molecule optical-trapping techniques to define the KIF15 mechanochemical cycle. We also studied the inhibitory effects of KIF15-IN-1, an uncharacterized, commercially available, small-molecule inhibitor, on KIF15 motility. To explore the complementary behaviors of KIF15 and Eg5, we also scored the effects of small-molecule inhibitors on admixtures of both motors, using both a microtubule (MT)-gliding assay and an assay for cancer cell viability. We found that (i) KIF15 motility differs significantly from Eg5; (ii) KIF15-IN-1 is a potent inhibitor of KIF15 motility; (iii) MT gliding powered by KIF15 and Eg5 only ceases when both motors are inhibited; and (iv) pairing KIF15-IN-1 with Eg5 inhibitors synergistically reduces cancer cell growth. Taken together, our results lend support to the notion that a combination drug therapy employing both inhibitors may be a viable strategy for overcoming chemotherapeutic resistance.

Keywords: filanesib; ispinesib; mitotic spindle; optical tweezers; single-molecule biophysics.

Fig. 1.

Optical force-clamp assay for KIF15. Polypeptide representations of KIF15 show motor domains (purple), coiled-coil regions (green) identified by COILS scores (43) (SI Appendix, Fig. S1), and a 6× His-tag (orange) of the full-length human KIF15 (A; 1,388 aa) and the truncated construct used in this study (B; 700 aa). (C) Histogram of measured KIF15 step sizes (n = 436) under a 4-pN hindering load in saturating ATP (2 mM ATP; error bars indicate counting errors), with superposed Gaussian (solid line) and fitted mean step size, d_step (mean ± SE). A schematic depicts the experimental geometry of the optical-trapping assay (not to scale), showing arrangements for hindering load (D; −) and assisting load (E; +); directions of kinesin motion (light arrows) and applied load (heavy arrows) are indicated. (F) Representative records of individual KIF15 trajectories obtained under hindering (−4 pN; blue) or assisting (+2 pN; purple) loads at saturating ATP. Median-filtered traces (dark colors; seven-point sliding window) are superposed on the unfiltered data (pale colors).

Fig. 2.

Single-molecule motility of KIF15 and Eg5 under load. Velocity (A) and randomness (B) are shown as functions of applied load under saturating ATP conditions (open circles; mean ± SE) for KIF15 (blue; n = 79–247) and Eg5 (red; n = 62–212). Velocity (C) and randomness (D) are shown as functions of the ATP concentration (open circles; mean ± SE) for KIF15 (blue; n = 78–247; F = −2 pN) and Eg5 (red; n = 22–92; F = 0 pN and F = −4 pN). Solid lines in all panels represent global fits to the four-state model of Fig. 3. Eg5 data (but not model fits) are replotted from a study by Valentine et al. (20).

Fig. 3.

Mechanochemical cycles of KIF15 and Eg5. (A) Minimal four-state kinetic representation of the kinesin mechanochemical cycle, based on a study by Milic et al. (50). (B) Transitions in the four-state scheme are assigned to specific biomechanical events by the partial NL-docking model. The first kinetic transition of this scheme, [1] → [2], represents reversible ATP binding to the nucleotide-free (Ø) MT-bound head of a 1-HB kinesin in the ATP-waiting state. This transition, associated with the rates for ATP binding ($k_1\left[\text{ATP}\right]$) and ATP release ($k_{-1}$), is followed by the single, load-dependent transition of the kinesin cycle, [2] → [3]. This mechanical transition, modeled by a force-dependent rate, $k_2\left(F\right)$, corresponds to partial docking of the NL to the MT-bound head. ATP hydrolysis then completes NL docking, which enables the tethered head to bind the MT, releasing ADP in the process, and ultimately advancing the dimer by ∼8 nm. In the minimal model, these events have been combined into a single transition, [3] → [4], associated with the rate constant, $k_3$. This composite transition implies passage through a 1-HB, posthydrolysis state, [3^‡], from which either (i) the tethered head binds the MT, [3^‡] → [4], thereby allowing the motor to continue stepping along the MT, or (ii) the bound head prematurely hydrolyzes ATP, leading to dissociation from the MT, [3^‡] → [4_off]. Finally, the trailing head undergoes transition [4] → [1], associated with the rate constant, $k_4$, which entails releasing P_i and detaching from the MT, thereby returning the dimer to the 1-HB ATP-waiting state. The four-state scheme also incorporates a back-stepping transition, $k_{\text{back}}$. In both panels, the nucleotide states of each head (purple/pink) are indicated as the dimer moves along the MT (green). ADP•P_i indicates heads in the posthydrolysis state. (C) Model parameters (rates and distances; mean ± SE) obtained by global fits to velocity and randomness measurements for KIF15 (blue) and Eg5 (red), based on the kinetic scheme in A (also Fig. 2 and SI Appendix, Fig. S2 A and B).

Fig. 4.

Single-molecule KIF15 and Eg5 processivity. (A) KIF15 RL (bars; mean ± SE; n = 47–144) under a +2-pN load in the presence of ATP or ATPγS at the indicated concentrations. No statistically significant change occurred in response to varying the ATP concentration or replacing it by ATPγS. (B) Measurements of RL as a function of applied load at saturating ATP (open circles; mean ± SE) for KIF15 (blue; n = 79–247) and Eg5 (red; n = 11–277). Linear fits (solid lines) are shown. The Eg5 data (but not the fits) are replotted from a study by Valentine and Block (54) (also SI Appendix, Fig. S2C.). (C) MT dissociation rates as functions of applied load (open circles; mean ± SE) for KIF15 (blue; n = 79–247) and Eg5 (red; n = 11–277). Dissociation rates as functions of load were modeled (solid lines) by dividing the fit values of the velocity (Fig. 2A) for KIF15 (blue) or Eg5 (red) by the fit values of the RL at the corresponding forces in B.

Fig. 5.

MT gliding by KIF15 and Eg5 in the presence of inhibitors. (A) Cartoon representation of the gliding assay (not to scale): MTs (green) are transported by kinesin motors (purple) attached via their stalks to a glass coverslip (blue) (Materials and Methods). The chemical structures of KIF15-IN-1 (B), ispinesib (C), and filanesib (D) are shown. (E) MT-gliding velocities (open circles; mean ± SE) generated by KIF15 in the presence of KIF15-IN-1 (blue; n = 14–31) and Eg5 in the presence of ispinesib (red; n = 9–30) or filanesib (dark red; n = 16–27) as functions of inhibitor concentration. Solid lines are exponential fits. (F) Table of IC₅₀ values (mean ± SE) from E. (G) MT-gliding velocities (mean ± SE) in the presence (shaded bars) or absence (open bars) of inhibitors, at the indicated concentrations, by Eg5 (red; n = 12–25), KIF15 (blue; n = 19–23), or both motors (purple; n = 16–35) (also SI Appendix, Fig. S3). (H) MT-gliding velocities (open circles; mean ± SE) generated by mixtures of motors. The KIF15-to-Eg5 motor ratio is expressed as the percentage of KIF15. Velocities were measured in the presence or absence of inhibitors at the concentrations indicated: no inhibitors (gray; n = 15–56), ispinesib (red; n = 12–52), filanesib (dark red; n = 21–37), KIF15-IN-1 (blue; n = 21–32), or KIF15-IN-1 with either ispinesib (light purple; n = 14–20) or filanesib (dark purple; n = 15–19). Sigmoidal (solid lines) and linear fits (dashed lines) are shown. Shaded backgrounds match the corresponding data in G and H, where inhibitor concentrations were chosen to be sufficient to fully inhibit the target motor, based on the data in E.

Fig. 6.

Cell growth under combined inhibition of KIF15 and Eg5, quantified by mCherry fluorescence. Synergistic effects of KIF15 and Eg5 inhibitors were investigated with a HeLa cell proliferation assay in the presence of KIF15-IN-1 and either ispinesib (A–E) or filanesib (F–J). (A and F) Number of viable HeLa cells after 72 h of treatment (open circles; mean ± SE; n = 3) with pairwise combinations of KIF15 and Eg5 inhibitors (Materials and Methods), reported as a percentage relative to the control (no inhibitor). (B and G) Cell proliferation data from A and F, presented as a grid displaying the observed inhibition for each pairwise combination of drug doses (note that 0% cell viability corresponds to 100% inhibition). (C and H) Expected level of inhibition for a pair of inhibitors that act independent of one another (Bliss independence; Materials and Methods). (D and I) Grid displaying numerical differences between the observed and expected levels of inhibition (assuming independence) for each pairwise combination of drug doses, where positive values indicate synergy. (E and J) Strongest enhancement in the level of inhibition of viable cell number (bars; mean ± SE; n = 3), beyond that expected for no synergy (dotted horizontal lines), occurred when 20 μM KIF15-IN-1 was paired with 1 nM ispinesib (E; ****P = 3 × 10⁻⁶; one-tailed t test) or with 1 nM filanesib (J; ****P = 8 × 10⁻⁵; one-tailed t test) (also SI Appendix, Fig. S4).