UA62784, a novel inhibitor of centromere protein E kinesin-like protein

Abstract

Pancreatic carcinoma is the fourth leading cause of death from cancer. Novel targets and therapeutic options are needed to aid in the treatment of pancreatic cancer. The compound UA62784 is a novel fluorenone with inhibitory activity against the centromere protein E (CENP-E) kinesin-like protein. UA62784 was isolated due to its selectivity in isogenic pancreatic carcinoma cell lines with a deletion of the DPC4 gene. UA62784 causes mitotic arrest by inhibiting chromosome congression at the metaphase plate likely through inhibition of the microtubule-associated ATPase activity of CENP-E. Furthermore, CENP-E binding to kinetochores during mitosis is not affected by UA62784, suggesting that the target lies within the motor domain of CENP-E. UA62784 is a novel specific inhibitor of CENP-E and its activity suggests a potential role for antimitotic drugs in treating pancreatic carcinomas.

Figure 1

Synthesis of UA62784

Figure 2

Flow cytometric cell cycle analysis following recovery from UA62784 treatment. MiaPaCa cells were treated with UA62784 for 8 hrs. and allowed to recover in fresh media for the indicated amount of time. The cells were harvested, incubated with PI and analyzed by flow cytometry. The results show a loss of G2/M accumulation (far right peak) and a gain of the G1 population (left peak) over time.

Figure 3

Mitotic index in cells treated with UA62784. Cells were incubated with the indicated amounts of UA62784 for 8 hours (a), Demecolcine for 24 hours (b), or Doxorubicin for 12 hours (c). Cells were harvested and incubated with an anti-phospho-Histone H3 antibody and propidium iodide (PI). Samples were analyzed by flow cytometry to quantify the mitotic index, indicated by a 4n DNA content and positive staining for phospho-Histone-H3. Error bars shown in (a) and (b) represent standard deviation (n=3). Asterisks indicate statistically significant results (p=0.05).

Figure 4

Kinesin ATPase assay using CENP-E purified motor domain protein. ATPase activity was measured by generation of Pi following addition of ATP and taxol-stabilized MTs to purified kinesin motor protein. Bkg- Background; no ATP added, No MTs- no microtubules added to reaction, DMSO-negative control, no UA62784 added. Error bars are shown to indicate standard deviation (n=4).

Figure 5

Immunofluorescent confocal microscopy (40X) staining for phospho-Histone H3 (red), a mitotic marker, and CENP-E (4). Panc-1 cells were left untreated (a-c) or were treated with 500nM UA62784 for 12 hrs. (d). Progression through mitosis is shown in a-c with CENP-E localizing diffusely in the cytoplasm during prophase (a), localizing to the DNA/kinetochores during prometaphase/metaphase (b), and localizing to the spindle midzone during anaphase (c). All cells in (a-c) are representative of one untreated sample. Note: only one cross-section of each confocal image is shown. These data are representative of 3 independent trials.

Figure 6

CENP-E microtubule-binding assay in the presence of UA62784. Purified taxol-stabilized microtubules were incubated with microtubule-associated protein extract (lanes 2-3), bovine serum albumin (4-5), CENP-E (6-7), or CENP-E and indicated concentrations of UA62784 (8-13) and ultracentrifuged to pellet microtubules. Any proteins that bind microtubules would be found primarily in the pellet (P) fraction whereas proteins that do not bind microtubules would be found primarily in the supernatant (S) fraction. Blots were visualized using IR secondaries (a) to indicate CENP-E and tubulin proteins only and Coomassie Blue staining (b) to indicate total protein. These data are representative of 4 independent experiments.