Fission yeast cells overproducing HSET/KIFC1 provides a useful tool for identification and evaluation of human kinesin-14 inhibitors

Abstract

Many cancer cells contain more than two centrosomes, yet these cancer cells can form bipolar spindles and appear to proliferate normally, instead of committing lethal mitoses with multipolar spindles. It is shown that extra centrosomes are clustered into two pseudo-bipolar spindle poles, thereby escaping from multipolarity. Human kinesin-14 (HSET or KIFC1), a minus end-directed motor, plays a crucial role in centrosome clustering and as such, HSET is essential for cell viability only in cancer cells with supernumerary centrosomes, but not in non-transformed cells. Accordingly, HSET is deemed to be an efficient chemotherapeutic target to selectively kill cancer cells. Recently, three HSET inhibitors (AZ82, CW069 and SR31527) have been reported, but their specificity, efficacy and off-target cytotoxicity have not been evaluated rigorously. Here we show that these inhibitors on their own are cytotoxic to fission yeast, suggesting that they have other targets in vivo except for kinesin-14. Nonetheless, intriguingly, AZ82 can neutralize overproduced HSET and partially rescue its lethality. This methodology of protein overproduction in fission yeast provides a convenient, functional assay system by which to screen for not only selective human kinesin-14 inhibitors but also those against other molecules of interest.

Modeling reveals cortical dynein-dependent fluctuations in bipolar spindle length

“…Biological inhibition or knockdown of Eg5 and overexpression of HSET are shown to alter spindle bipolarity (6,30,65,66,(81)(82)(83). We manipulate motor activity in the model by perturbing the motor-MT binding probability, which accurately captures spindle collapse with no Eg5 or HSET activity (Fig.…”

“…Biological results also show that high HSET activity increases the frequency of monopolar spindles (81)(82)(83). To test that our model accurately reflects this role of HSET activity, we mimic HSET overexpression by setting the probability of binding to MTs (P H ) equal to one.…”

Computational benchmarking of putative KIFC1 inhibitors

“…Specifically, AZ82 decreased the viability of yeast cells despite the absence of KIFC1 in the cells. 65 However, no morphological changes were detected. Interestingly, AZ82 at 10 μM rescued cell death mediated by KIFC1 overexpression in yeast cells, further validating its binding to KIFC1 even though nonspecifically.…”

“…The 1,2,4-thiadiazole derivative SR31527 (Figure 7) was identified as a KIFC1 inhibitor that targets the microtubulestimulated ATPase activity of KIFC1. 65 Among other HTS hits, SR31527 had the most potent activity (IC 50 : 6.6 μM) and favorable structural features. To assess whether SR31527 binds KIFC1 only or the complex KIFC1microtubules, Zhang et al performed a binding assay using biolayer interferometry and found that SR31527 displayed direct binding to KIFC1 with a K d of 25.4 nM.…”

“…Interestingly, AZ82 at 10 μM rescued cell death mediated by KIFC1 overexpression in yeast cells, further validating its binding to KIFC1 even though nonspecifically. 65 The precise mode of binding of AZ82 to KIFC1 has also been explored. Based on its similarity to other kinesin inhibitors, AZ82 was initially proposed to bind to the L5/α2/α3 binding site of KIFC1.…”