A screen for kinetochore-microtubule interaction inhibitors identifies novel antitubulin compounds

Abstract

Background: Protein assemblies named kinetochores bind sister chromatids to the mitotic spindle and orchestrate sister chromatid segregation. Interference with kinetochore activity triggers a spindle checkpoint mediated arrest in mitosis, which frequently ends in cell death. We set out to identify small compounds that inhibit kinetochore-microtubule binding for use in kinetochore-spindle interaction studies and to develop them into novel anticancer drugs.

Methodology/principal findings: A fluorescence microscopy-based in vitro assay was developed to screen compound libraries for molecules that prevented the binding of a recombinant human Ndc80 kinetochore complex to taxol-stabilized microtubules. An active compound was identified that acted at the microtubule level. More specifically, by localizing to the colchicine-binding site in alphabeta-tubulin the hit compound prevented the Ndc80 complex from binding to the microtubule surface. Next, structure-activity analyses distinguished active regions in the compound and led to the identification of highly potent analogs that killed cancer cells with an efficacy equaling that of established spindle drugs.

Conclusions/significance: The compound identified in our screen and its subsequently identified analogs represent new antitubulin chemotypes that can be synthetically developed into a novel class of antimitotic spindle drugs. In addition, they are stereochemically unique as their R- and S-isomers mimic binding of colchicine and podophyllotoxin, respectively, two antitubulin drugs that interact differently with the tubulin interface. Model-driven manipulation of our compounds promises to advance insight into how antitubulin drugs act upon tubulin. These advances in turn may lead to tailor-made colchicine site agents which would be valuable new assets to fight a variety of tumors, including those that have become resistant to the (antispindle) drugs used today.

Figure 1. Screen for compounds inhibiting binding of the recombinant Ndc80 kinetochore complex to taxol-stabilized microtubules.

Schematic of the expression, purification and fluorescent labeling of the recombinant human Ndc80 kinetochore complex (Ndc80⁴⁸⁸ complex) used in the screen (A). Recombinant Ndc80 complex obtained by GST-based purification and PreScission Protease-mediated release from the affinity beads (left panel). Gel filtration profile (Superdex 200) of the Ndc80⁴⁸⁸ complex obtained by labeling the purified complex with Alexa Fluor 488 C₅-maleimide fluorophore (right panel) (B). Schematic of the fluorescence microscopy-based screen for compounds inhibiting the binding of the Ndc80⁴⁸⁸ complex (green) to rhodamine (red) labeled, taxol-stabilized MTs (tubulin subunits are colored black and grey) (C).

Figure 2. Compound B prevents the recombinant Ndc80 kinetochore complex from binding to taxol-stabilized microtubules.

Effect of compound B (50 µmol/l) on binding of the Ndc80⁴⁸⁸ complex to rhodamine-labeled, taxol-stabilized MTs as assayed using inverted wide-field fluorescence microscopy. The scale bar represents 4 µm (A). Structure of compound B; 6-furan-2-yl-3-methyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-2-carboxylic acid (tetrahydro-furan-2-yl)methyl ester (B). Measurement of the IC₅₀ value of compound B using total internal reflection fluorescence microscopy. The scale bar represents 4 µm (C). Compound B (50 µmol/l) prevented MT-associating protein CLIP-170⁴⁸⁸, used as a specificity control, from binding to taxol-stabilized MTs. The scale bar represents 4 µm (D). Antitubulin control drug nocodazole (50 µmol/l) did not prevent the Ndc80⁴⁸⁸ complex from binding to MTs. The scale bar represents 4 µm (E).

Figure 3. Compound B inhibits tubulin assembly and destabilizes spindles, resulting in mitotic block and cell death.

Live-cell imaging of HeLa cells synchronized in G1/S and released into medium containing compound B (50 µmol/l) or mock solution (1% DMSO). The scale bar represents 20 µm. The plot shows the mitotic index of the cultures over time (compound B, red bars; mock [1% DMSO], green bars) (A). Compound B induced a mitotic delay (1–10 µmol/l) and cell death (5–10 µmol/l) (B). The mitotic delay triggered by compound B (10 µmol/l) resulted from mitotic checkpoint activity as indicated by the presence of SAC protein Mad1 at kinetochores. Nocodazole (0.5 µmol/l) acted as a positive control. The scale bar represents 10 µm (C). Compound B inhibited spindle formation (row 2) and destabilized metaphase spindles (row 5) in HeLa cells. Nocodazole (0.5 µmol/l) acted as the positive control. The scale bar represents 10 µm (D). The antitubulin activity of compound B on human cells is reversible as the cells formed a mitotic spindle structure when the drug was washed out and the cells were released into fresh medium containing 10 µmol/l MG132. Nocodazole (0.5 µmol/l) acted as the positive control. The scale bar represents 10 µm (E).

Figure 4. Taxol inhibits the microtubule-destabilizing activity of compound B.

Taxol (0.5 µmol/l) prevented spindle depolymerization by compound B (10 µmol/l) when both were added to HeLa cells arrested in metaphase with proteosome inhibitor MG132. The scale bar represents 10 µm (A). Depolymerization by compound B, nocodazole, and maytansine (50 µmol/l each) of taxol-stabilized MTs in vitro. The bars represent standard errors (B).

Figure 5. Compound B inhibits tubulin assembly and destabilizes MTs by acting at the tubulin colchicine site.

Compound B inhibited tubulin assembly in vitro; combretastatin A-4 and nocodazole acted as positive controls (A). Compound B depolymerized non-stabilized MTs in vitro; nocodazole acted as the positive control (B). Compound B competed with [³H]colchicine for binding to the colchicine site in αβ-tubulin. The bars represent standard deviations (C).

Figure 6. Furan-metoticas inhibit tubulin assembly and destabilize microtubules.

Structures of furan-metoticas B, A6, D7, E7, and A8, and their IC₅₀ values measured in the Ndc80⁴⁸⁸-MT binding assay (A). Compounds A6, D7, E7 and A8 inhibited tubulin polymerization in vitro with an efficiency exceeding that of compound B; combretastatin A-4 and nocodazole acted as positive controls. The bars represent standard deviations (B). Compounds A6, D7, E7 and A8 efficiently depolymerized non-stabilized MTs in vitro; nocodazole acted as a positive control (C). Compounds B, A6, D7, E7 and A8 competed with [³H]colchicine for binding to tubulin; combretastatin A-4 acted as the positive control. The bars represent standard deviations (D). Furan-metoticas triggered a mitotic block and subsequent cell death with high efficiency in HeLa cells (cervical adenocarcinoma). The bars represent standard deviations (E).

Figure 7. Compound A8 inhibits tubulin assembly and destabilizes spindle microtubules in HeLa cells and in vitro.

Live-cell videomicroscopy of HeLa cells treated with mock solution (1% DMSO) or with compound A8 (0.5 µmol/l) following release from synchronization in G1/S (upper panel). The scale bar represents 20 µm. The plot shows the mitotic index of the cell cultures over time (compound A8, red bars; mock [1% DMSO], green bars) (A). Compound A8 inhibited spindle formation (α-tubulin) in HeLa cells characterized by detached sister chromatids (DAPI/CREST staining), as shown by confocal IF microscopy; nocodazole acted as a positive control. The antitubulin activity of compound A8 on human cells is reversible as the cells formed a mitotic spindle structure when the drug was washed out and the cells were released into fresh medium containing 10 µmol/l MG132. The scale bar represents 10 µm (B). Compound A8 inhibited tubulin assembly (upper panel) and triggered MT depolymerization in vitro (lower panel) (C). Compound A8 (50 µmol/l) mildly depolymerized taxol-stabilized MTs in vitro; nocodazole and maytansine (50 µmol/l) acted as positive controls. The bars represent standard errors (D).

Figure 8. Furan-metoticas kill proliferating cells with an efficiency equaling that of established antitubulin drugs.

Viability analysis of hTERT RPE-1 control cells and two adenocarcinoma cell lines following treatment with compound A8 (1–1000 nmol/l); nocodazole and colchicine acted as positive controls.