Caspase activity is not required for the mitotic checkpoint or mitotic slippage in human cells

Biochemical studies suggest that caspase activity is required for a functional mitotic checkpoint (MC) and mitotic slippage. To test this directly, we followed nontransformed human telomerase immortalized human retinal pigment epithelia (RPE-1) cells through mitosis after inhibiting or depleting selected caspases. We found that inhibiting caspases individually, in combination, or in toto did not affect the duration or fidelity of mitosis in otherwise untreated cells. When satisfaction of the MC was prevented with 500 nM nocodazole or 2.5 μM dimethylenastron (an Eg5 inhibitor), 92-100% of RPE-1 cells slipped from mitosis in the presence of pan-caspase inhibitors or after simultaneously depleting caspase-3 and -9, and they did so with the same kinetics (~21-22 h) as after treatment with nocodazole or Eg5 inhibitors alone. Surprisingly, inhibiting or depleting caspase-9 alone doubled the number of nocodazole-treated, but not Eg5-inhibited, cells that died in mitosis. In addition, inhibiting or depleting caspase-9 and -3 together accelerated the rate of slippage ~40% (to ~13-15 h). Finally, nocodazole-treated cells that recently slipped through mitosis in the presence or absence of pan-caspase inhibitors contained numerous BubR1 foci in their nuclei. From these data, we conclude that caspase activity is not required for a functional MC or for mitotic slippage.     

Identification of the protein binding region of S-trityl-L-cysteine, a new potent inhibitor of the mitotic kinesin Eg5

Human Eg5, a mitotic motor of the kinesin superfamily, is involved in the formation and maintenance of the mitotic spindle. The recent discovery of small molecules that inhibit HsEg5 by binding to its catalytic motor domain leading to mitotic arrest has attracted more interest in Eg5 as a potential anticancer drug target. We have used hydrogen-deuterium exchange mass spectrometry and directed mutagenesis to identify the secondary structure elements that form the binding sites of new Eg5 inhibitors, in particular for S-trityl-l-cysteine, a potent inhibitor of Eg5 activity in vitro and in cell-based assays. The binding of this inhibitor modifies the deuterium incorporation rate of eight peptides that define two areas within the motor domain: Tyr125-Glu145 and Ile202-Leu227. Replacement of the Tyr125-Glu145 region with the equivalent region in the Neurospora crassa conventional kinesin heavy chain prevents the inhibition of the Eg5 ATPase activity by S-trityl-l-cysteine. We show here that S-trityl-l-cysteine and monastrol both bind to the same region on Eg5 by induced fit in a pocket formed by helix alpha3-strand beta5 and loop L5-helix alpha2, and both inhibitors trigger similar local conformational changes within the interaction site. It is likely that S-trityl-l-cysteine and monastrol inhibit HsEg5 by a similar mechanism. The common inhibitor binding region appears to represent a "hot spot" for HsEg5 that could be exploited for further inhibitor screening.     

Mitotic kinesin inhibitors induce mitotic arrest and cell death in Taxol-resistant and -sensitive cancer cells

Taxanes are powerful chemotherapy agents that target the microtubule cytoskeleton, leading to mitotic arrest and cell death; however, their clinical efficacy has been hampered due to the development of drug resistance. Therefore, other proteins involved in spindle assembly are being examined as potential targets for anticancer therapy. The mitotic kinesin, Eg5 is critical for proper spindle assembly; as such, inhibition of Eg5 leads to mitotic arrest making it a potential anticancer target. We wanted to validate Eg5 as a therapeutic target and determine if Eg5 inhibitors retain activity in Taxol-resistant cells. Using affinity chromatography we first show that the compound HR22C16 is an Eg5 inhibitor and does not interact with other microtubule motor proteins tested. Furthermore, HR22C16 along with its analogs, inhibit cell survival in both Taxol-sensitive and -resistant ovarian cancer cells with at least 15-fold greater efficacy than monastrol, the first generation Eg5 inhibitor. Further analysis with HR22C16-A1, the most potent HR22C16 analog, showed that it retains efficacy in PgP-overexpressing cells, suggesting that it is not a PgP substrate. We further show that HR22C16-A1 induces cell death following mitotic arrest via the intrinsic apoptotic pathway. Interestingly, the combination of HR22C16-A1 with Taxol results in an antagonistic antiproliferative and antimitotic effect, possibly due to the abrogation of Taxol-induced mitotic spindles by HR22C16-A1. Taken together, our results show that Eg5 inhibitors have promising anticancer activity and can be potentially used to overcome Taxol resistance in the clinical setting.     

Crystal structure of HsEg5 in complex with clinical candidate CK0238273 provides insight into inhibitory mechanism, potency, and specificity

HsEg5 is an important mitotic kinesin responsible for bipolar spindle formation at early mitosis. A rich body of evidence shows that inhibition of HsEg5 can result in mitotic arrest followed by cellular apoptosis. Recently identified HsEg5 inhibitor, CK0238273, exhibits potent antitumor activity and is currently in clinical trial. Here we report the cocrystal structure of the motor domain of HsEg5 in complex with CK0238273 at a 2.15 Å resolution. Compared to the previously published HsEg5-Monastrol complex structure, CK0238273 shares the same induced-fit pocket with similar allosteric inhibitory mechanism. However, CK0238273 shows better fitting to the binding pocket with 65% increase of hydrophobic interaction area than that of Monastrol. Some unique hydrophilic interactions were also observed mostly between the phenyl ring and 8-chloro on quinazolinone of CK0238273 with ARG221 and GLY217. We believe that the combination of these interactions defines the superior potency and specificity of CK0238273.     

New chemical tools for investigating human mitotic kinesin Eg5

We have designed and synthesized a series of monastrol derivatives, an allosteric inhibitor of Eg5, a motor protein responsible for the formation and maintenance of the bipolar spindle in mitotic cells. Sterically demanding structural modifications have been introduced on the skeleton of the parent drug either via a multicomponent Biginelli reaction or a stepwise modification of monastrol. The ability of these compounds to inhibit Eg5 activity has been investigated using two in vitro steady-state ATPase assays (basal and microtubule-stimulated) as well as a cell-based assay. One compound in the series appeared more potent than monastrol by a fivefold factor. Three other compounds that were unable to inhibit Eg5 ATPase activity in vitro proved potent Eg5 inhibitors in the cell-based assay. The results obtained led to the identification of structure-activity relationships further used to design an affinity matrix that can be used for fast and efficient purification of Eg5 from crude lysate of eukaryotic cells.     

Synthesis and biological evaluation of new tetrahydro-beta-carbolines as inhibitors of the mitotic kinesin Eg5

The mitotic kinesin Eg5 (or KSP) is a crucial player in the development and function of the mitotic spindle. Inhibition of this protein leads to cell cycle arrest and apoptosis without interfering with other microtubule-dependent processes. Therefore, it is a potential target in cancer therapy. Here, we report the synthesis and biological evaluation of a small library of molecules based on the structure of the known Eg5 inhibitor HR22C16. One of these derivatives (compound trans-24) proved to be a potent and specific Eg5 inhibitor.     

S-trityl-L-cysteine is a reversible, tight binding inhibitor of the human kinesin Eg5 that specifically blocks mitotic progression

Human Eg5, responsible for the formation of the bipolar mitotic spindle, has been identified recently as one of the targets of S-trityl-L-cysteine, a potent tumor growth inhibitor in the NCI 60 tumor cell line screen. Here we show that in cell-based assays S-trityl-L-cysteine does not prevent cell cycle progression at the S or G(2) phases but inhibits both separation of the duplicated centrosomes and bipolar spindle formation, thereby blocking cells specifically in the M phase of the cell cycle with monoastral spindles. Following removal of S-trityl-L-cysteine, mitotically arrested cells exit mitosis normally. In vitro, S-trityl-L-cysteine targets the catalytic domain of Eg5 and inhibits Eg5 basal and microtubule-activated ATPase activity as well as mant-ADP release. S-trityl-L-cysteine is a tight binding inhibitor (estimation of K(i,app) <150 nm at 300 mm NaCl and 600 nm at 25 mm KCl). S-trityl-L-cysteine binds more tightly than monastrol because it has both an approximately 8-fold faster association rate and approximately 4-fold slower release rate (6.1 microM(-1) s(-1) and 3.6 s(-1) for S-trityl-L-cysteine versus 0.78 microM(-1) s(-1) and 15 s(-1) for monastrol). S-trityl-L-cysteine inhibits Eg5-driven microtubule sliding velocity in a reversible fashion with an IC(50) of 500 nm. The S and D-enantiomers of S-tritylcysteine are nearly equally potent, indicating that there is no significant stereospecificity. Among nine different human kinesins tested, S-trityl-L-cysteine is specific for Eg5. The results presented here together with the proven effect on human tumor cell line growth make S-trityl-L-cysteine a very attractive starting point for the development of more potent mitotic inhibitors.     

Structure-activity relationship of pyrazolo pyrimidine derivatives as inhibitors of mitotic kinesin Eg5 and anticancer agents

Human kinesin Eg5 is a potential inhibiting site for cancer chemotherapy. Blocking metaphase by binding foreign inhibitors with Eg5 eventually leads to apoptotic cell death. Here, we report the pyrazolopyrimidine derivates as potent inhibitors of Eg5 that prevents mitotic kinesin progression. IC₅₀ values were evaluated against the motor domain of Eg5 using steady-state ATPase assay. To better understanding, we have performed molecular docking simulation. It reveals that the interactions of the proposed inhibitors with both the allosteric sites (helices α2, α3 and loopL5, and helices α4 & α6). Out of fifteen pyrazolopyrimidine derivates, three compounds (12, 25, and 27) have shown significant inhibition of Eg5. The synthesized compounds (12, 25, and 27) were tested for their in-vitro anticancer activity against cervical cancer cell line (HeLa).     

Kinetochore-microtubule stability governs the metaphase requirement for Eg5

The mitotic spindle is a bipolar, microtubule (MT)-based cellular machine that segregates the duplicated genome into two daughter cells. The kinesin-5 Eg5 establishes the bipolar geometry of the mitotic spindle, but previous work in mammalian cells suggested that this motor is unimportant for the maintenance of spindle bipolarity. Although it is known that Kif15, a second mitotic kinesin, enforces spindle bipolarity in the absence of Eg5, how Kif15 functions in this capacity and/or whether other biochemical or physical properties of the spindle promote its bipolarity have been poorly studied. Here we report that not all human cell lines can efficiently maintain bipolarity without Eg5, despite their expressing Kif15. We show that the stability of chromosome-attached kinetochore-MTs (K-MTs) is important for bipolar spindle maintenance without Eg5. Cells that efficiently maintain bipolar spindles without Eg5 have more stable K-MTs than those that collapse without Eg5. Consistent with this observation, artificial destabilization of K-MTs promotes spindle collapse without Eg5, whereas stabilizing K-MTs improves bipolar spindle maintenance without Eg5. Our findings suggest that either rapid K-MT turnover pulls poles inward or slow K-MT turnover allows for greater resistance to inward-directed forces.     

Eg5 is static in bipolar spindles relative to tubulin: evidence for a static spindle matrix

We used fluorescent speckle microscopy to probe the dynamics of the mitotic kinesin Eg5 in Xenopus extract spindles, and compared them to microtubule dynamics. We found significant populations of Eg5 that were static over several seconds while microtubules flux towards spindle poles. Eg5 dynamics are frozen by adenylimidodiphosphate. Bulk turnover experiments showed that Eg5 can exchange between the spindle and the extract with a half life of <55 s. Eg5 distribution in spindles was not perturbed by inhibition of its motor activity with monastrol, but was perturbed by inhibition of dynactin with p50 dynamitin. We interpret these data as revealing the existence of a static spindle matrix that promotes Eg5 targeting to spindles, and transient immobilization of Eg5 within spindles. We discuss alternative interpretations of the Eg5 dynamics we observe, ideas for the biochemical nature of a spindle matrix, and implications for Eg5 function.