Synergistic blockade of mitotic exit by two chemical inhibitors of the APC/C

Abstract

Protein machines are multi-subunit protein complexes that orchestrate highly regulated biochemical tasks. An example is the anaphase-promoting complex/cyclosome (APC/C), a 13-subunit ubiquitin ligase that initiates the metaphase–anaphase transition and mitotic exit by targeting proteins such as securin and cyclin B1 for ubiquitin-dependent destruction by the proteasome1,2. Because blocking mitotic exit is an effective approach for inducing tumour cell death3,4, the APC/C represents a potential novel target for cancer therapy. APC/C activation in mitosis requires binding of Cdc20 (ref. 5), which forms a co-receptor with the APC/C to recognize substrates containing a destruction box (D-box)6,7,8,9,10,11,12,13,14. Here we demonstrate that we can synergistically inhibit APC/C-dependent proteolysis and mitotic exit by simultaneously disrupting two protein–protein interactions within the APC/C–Cdc20–substrate ternary complex. We identify a small molecule, called apcin (APC inhibitor), which binds to Cdc20 and competitively inhibits the ubiquitylation of D-box-containing substrates. Analysis of the crystal structure of the apcin–Cdc20 complex suggests that apcin occupies the D-box-binding pocket on the side face of the WD40-domain. The ability of apcin to block mitotic exit is synergistically amplified by co-addition of tosyl-l-arginine methyl ester, a small molecule that blocks the APC/C–Cdc20 interaction15,16. This work suggests that simultaneous disruption of multiple, weak protein–protein interactions is an effective approach for inactivating a protein machine.

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Figure 1: Apcin binds to Cdc20 and inhibits APC/C-dependent ubiquitylation.
Figure 2: Apcin binds to the D-box binding site of Cdc20.
Figure 3: Effects of apcin on Cdc20 binding to APC/C and stability of APC/C substrates in mitotic Xenopus extract.
Figure 4: Apcin synergizes with proTAME to prolong mitotic duration.

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Protein Data Bank

Data deposits

Structure coordinates have been deposited in Protein Data Bank under accession number 4N14.

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Acknowledgements

We thank W. Harper for providing constructs for WD40-containing proteins, T. Gahman for assistance with apcin synthesis and D. Tomchick for assistance with structure refinement. Results shown in this report are derived from work performed at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. This work was supported by grants from the National Institutes of Health (GM085004 to X.L. and GM066492 to R.W.K.) and by a grant from the Lynch Foundation to R.W.K.

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Contributions

K.L.S. and X.Z. performed experiments to identify Cdc20 as the target of apcin. K.L.S. characterized binding of apcin to Cdc20, and evaluated effects of Cdc20 mutations on apcin binding and cyclin proteolysis. W.T. purified Cdc20 and performed crystallization and structure determination of the Cdc20–apcin complex. H.Y. and X.L. contributed to structure determination and data analysis. N.D. and X.Z. characterized effects of apcin and TAME on substrate degradation, ubiquitylation and Cdc20 binding to APC/C in Xenopus extract. N.D. evaluated binding of apcin to Cdc20 using the thermal shift assay. M.Z. characterized effects of apcin and proTAME on mitotic index in fixed cell assays. K.L.S. and X.Z. characterized effects of apcin and proTAME by live cell imaging. F.S. developed the high-throughput mitotic index assay. K.L.S., X.Z., J.M., K.L.P. and F.S. analysed time-lapse videos. T.B.S. developed statistical models and performed statistical analysis. R.W.K. conceived the project, assisted with experimental design and data analysis, and wrote the manuscript, with assistance from all authors.

Corresponding author

Correspondence to Randall W. King.

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There is a patent application on this work, filed by Harvard University on behalf of the authors.

Extended data figures and tables

Extended Data Figure 1 Apcin binds Cdc20 and inhibits APC/C-dependent proteolysis in Xenopus extract.

a, Effects of apcin and derivatives (200 μM) on degradation of a cyclin B1 N-terminal fragment (cycB1-NT) in mitotic Xenopus egg extracts. Substrate was expressed in reticulocyte lysate and labelled with [35S]methionine. Samples were analysed by SDS-gel electrophoresis and phosphorimaging. Quantitation of the 40-min time point from three independent experiments is shown in Fig. 1b. b, Apcin-M and TAME do not inhibit binding of Cdc20 to apcin-A resin. The experiment was performed as shown in Fig. 1e except that the inactive apcin derivative apcin-M or the Cdc20-IR tail antagonist TAME were also tested and [35S]Cdc20 was detected by autoradiography. c, Apcin-A interacts strongly with Cdc20, weakly with Cdh1, and shows little interaction with other WD40-domain proteins. Proteins were expressed in reticulocyte lysate and labelled with [35S]methionine. Left panel, mean value for the percentage bound on the basis of three experiments (error bars, s.e.m.). Right panel, representative autoradiograph of one of three experiments. b, bound; i, 5% input; c, bound to empty resin (control). d, Apcin inhibits degradation of cycB1-NT in Cdh1-treated interphase extract. The addition of roscovitine (75 μM) is required to inhibit Cdk1 activity that suppresses Cdh1-dependent proteolysis. Note that 100 μM apcin is less effective at stabilizing the substrate in Cdh1-activated interphase extract than in mitotic Xenopus extract (Fig. 3b). e, Apcin binds to endogenous Cdc20 in Xenopus extract as measured by a thermal shift assay33. Extract was incubated with varying concentrations of apcin, heated to 46 °C for 3 minutes, and precipitated proteins removed by centrifugation. The soluble fraction was analysed by SDS–PAGE and western blotting for Cdc20. The left panel shows the percentage of soluble Cdc20 (mean ± s.e.m. from three independent experiments). Western blot from one of three experiments is shown below. For comparison, total Cdc20 from interphase extract (and various dilutions) is shown. Right panel, Coomassie-stained gel of soluble proteins, indicating that there is not an observable non-specific stabilization of proteins induced by apcin addition.

Extended Data Figure 2 Apcin acts as a competitive inhibitor of APC/C-dependent ubiquitylation.

a, Apcin inhibits formation of high-molecular-mass ubiquitin conjugates of full-length cyclin B1. Mitotic Xenopus extract was pre-treated with the deubiquitinating enzyme inhibitor ubiquitin vinyl sulfone (UbVS, 20 μM) and proteasome inhibitor MG262 (150 μM) to stabilize ubiquitin conjugates. [35S]cyclin B1 was added together with ubiquitin (44 μM) and samples analysed by SDS–PAGE and phosphorimaging. Right panel shows quantitation of the experiment. b, Apcin acts as a competitive inhibitor of APC/C-dependent ubiquitylation. APC/C was purified from mitotic Xenopus extracts and the initial rates of ubiquitylation of HA-tagged cycB1-NT were measured in the presence of methylated ubiquitin to prevent ubiquitin chain elongation. The reaction was stopped at 45 s and the products were detected by anti-HA blot. The left panel shows the anti-HA blot from one experiment; substrate concentrations were 62.5, 125, 250, 500 and 1,000 nM (left to right). Asterisk indicates an SDS-resistant aggregated form of substrate. Quantitation of three independent experiments, and a summary of kinetic parameters, are shown. Note that the effects of apcin in this reconstituted assay performed under initial rate conditions appear distinct from those obtained in crude Xenopus extract. See Supplementary Discussion for a more detailed discussion of these differences.

Extended Data Figure 3 Structure of the apcin-Cdc20 complex.

a, The Fo − Fc omit electron density map at the contour level of 3σ is shown. The density for the bound apcin conformation is unambiguous and is consistent with the structure–activity analysis and mutagenesis experiments. b, Overlay of the structure of the Cdc20–apcin structure with the structure of a D-box-containing protein bound to Cdh1 (ref. 14). The trichloromethyl group of apcin projects into a hydrophobic pocket that is occupied by the leucine of the RXXL motif of the D-box. The position of the arginine from the RXXL motif suggests a role for E465 of Cdc20 in a charge-based interaction with the D-box, consistent with our data that the E465S mutation disrupts the ability of Cdc20 to promote substrate degradation more than it perturbs apcin binding. c, Depletion with anti-Cdc20 antibody covalently coupled to protein A beads depletes endogenous Xenopus Cdc20 from mitotic extract. d, Example autoradiogram of data shown in Fig. 2b.

Extended Data Figure 4 Effects of apcin on Cdc20 binding to APC/C and stability of APC/C substrates in mitotic Xenopus extract.

a, Example western blot for data shown in Fig. 3a. b, Substrate-mediated recruitment of Cdc20 to APC/C in Xenopus extract is dependent on the D-box motif. Increasing concentrations of wild-type or different D-box mutants of cycB1-NT were introduced into mitotically arrested Xenopus extract and the APC/C was isolated with anti-Cdc27 antibodies by immunoprecipitation for 1 h at 4 °C. The immunoprecipitate was separated by SDS–PAGE and analysed by western blotting against Cdc20 and Cdc27. Levels of Cdc20 were quantitated using ImageJ and normalized to APC/C subunit Cdc27. c, Analysis of effects of apcin and TAME on APC/C substrate degradation in mitotic Xenopus extract. Levels of 35S-labelled substrates were assessed by SDS–PAGE and phosphorimaging. Asterisk represents a non-specific band. Images show one of three experiments quantitated in Fig. 3b. d, Experiment performed as in c, but examining the combined effects of apcin and TAME. Image shows one of three experiments quantitated in Fig. 3c.

Extended Data Figure 5 Effects of apcin and TAME on stability of APC/C substrates in mitotic Xenopus extract.

Apcin and TAME synergize in stabilizing cyclin A2 (a) and securin (b) in mitotic Xenopus extract. Levels of 35S-labelled substrates were assessed by SDS–PAGE and phosphorimaging. The change in mobility of securin between 0 and 20 min is probably a result of mitotic phosphorylation. Error bars in a represent mean and s.e.m. of three independent experiments. Data in b are representative of two independent experiments.

Extended Data Figure 6 Apcin has differential effects on substrate ubiquitylation in mitotic Xenopus extract that correlate with effects on proteolysis.

To examine the profile of ubiquitylated species generated in Xenopus extract, deubiquitylation and proteasome-mediated degradation were inhibited by pre-treatment with the general deubiquitinating-enzyme inhibitor ubiquitin-vinyl sulfone (UbVS; 20 μM), as established previously34, and the proteasome-inhibitor MG262 (150 μM). Next, wild-type ubiquitin (Ub; 44 μM) and apcin (100 μM) or DMSO were added. 35S-labelled APC/C substrates expressed in reticulocyte lysate were introduced into treated extract and their ubiquitylation at indicated times assessed by SDS–PAGE and phosphor imaging. Levels of substrates that were not modified with ubiquitin (unconjugated), modified with one to three ubiquitins (substrate-Ub, Ub ≤ 3) or modified with more than three ubiquitin moieties (substrate-Ub, Ub > 3) were quantitated by phosphorimaging and plotted relative to radiolabelled protein in the respective region of the gel at 0 min in the apcin or DMSO sample. a, cycB1-NT; b, cyclin B1; c Nek2A; d, cyclin A2; e, securin. Note that for cyclin A2, apcin reduces the amount of Ub conjugates with very high molecular mass (as indicated by inspection of the gel image) but does not reduce the fraction of conjugates modified with more than three ubiquitins (as indicated by the quantitation). These results are consistent with the inability of apcin to stabilize cyclin A2 in proteolysis assays, given that the proteasome typically requires at least four ubiquitin molecules to be attached to a substrate for efficient proteolysis.

Extended Data Figure 7 Apcin and proTAME synergize to block mitotic exit in human cells, as measured in a fixed cell assay.

a, Summary of the fixed-cell imaging assay and data processing methods to determine synergy between apcin and proTAME. See methods for detailed description of the assay. b, First column: primary data plotted as a heat map displaying mean fraction below threshold for each drug treatment concentration in each of four cell lines. This threshold is established on the basis of the mitotic index of DMSO-treated cells. Note that a high value in this column indicates a low mitotic index. Effects of single drugs alone are highlighted in pale purple. Second column (labelled ‘Synergy model’): calculated effect of the combination of drugs on the basis of a model that permits synergistic interaction between proTAME and apcin. Because this panel shows calculated values of combination effects, the effects of individual drugs are not shown. Note that the synergy model closely parallels the actual data shown in the first column. Third column: calculated effect of the combination of drugs on the basis of a model that permits only multiplicative interaction between proTAME and apcin (Bliss model). Because this panel shows calculated values of combination effects, the effects of individual drugs are not shown. Note that the Bliss model does not closely parallel the actual data shown in the first column. Fourth column: heat map of the difference between the synergy and Bliss model predictions shows the degree of synergy at each drug dose combination (same as Fig. 4a for RPE1 cells). *P < 0.05 for analysis of four technical replicates. See Supplementary Information for details of the statistical analysis. c, Activity of apcin derivatives in the fixed cell assay described in a, using RPE1 cells. Source data

Extended Data Figure 8 Apcin and proTAME synergize to block mitotic exit in human cells, as measured in a live cell assay.

a, Western blot of Mad2 knockdown in RPE1 cells by siRNA from one of the experiments shown in Fig. 4b. b, Analysis of cell fate in RPE1 cells for the experiment shown in Fig. 4b. c, Combined data from two independent experiments in RPE1 cells shown in Fig. 4b. ‘Cells’ is the total number of cells analysed, while ‘Fates observed’ is the subset of cells whose fate was observed, excluding cells that migrated out of view during the movie or were still arrested at the end of imaging. The median is the time on the x axis of the Kaplan–Meier curve corresponding to 0.5 on the y axis ‘Fraction of cells in mitosis’. d, Statistical modelling of data from experiment in Fig. 4b (RPE1 cells). The two tables on the left show the rate of mitotic exit relative to DMSO control for each of two siRNA treatment subgroups (control siRNA, left; Mad2 siRNA, middle), the P value for the comparison to DMSO (Cox proportional hazards model, see Supplementary Methods) and the P value for the comparison of proTAME with or without apcin-M. To determine P values for the pairwise comparisons of proTAME versus apcin-M with proTAME, we fitted a Cox proportional hazards model similar to that described in the Methods but with just an apcin-M effect and analysed just the subset of cells that were treated with either proTAME only or apcin-M with proTAME. The table labelled ‘Synergy between apcin and proTAME’ shows the rate of mitotic exit with both compounds (apcin with proTAME), relative to what would be predicted by a multiplicative combination of the effects of each compound alone. The magnitude of the synergy is roughly doubled when checkpoint activity is reduced by Mad2 siRNA. e, Synchronized U2OS H2B–GFP cells were treated with apcin or apcin-M (25 µM) and/or proTAME (12 µM). Cells were then imaged every 6 or 10 min for 45 h. Mitotic duration and cell fate were determined by manual inspection of the videos and plotted as Kaplan–Meier curves. The hatch marks on the Kaplan–Meier curves indicate mitotic duration endpoints of censored cells. Graphs include the combined results of five independent experiments. The U2OS model differs from the model used to test RPE1 data in that the U2OS analysis is not stratified by either date or person, and the U2OS data do not include an effect of apcin-M alone (in the absence of proTAME). Pairwise comparison between proTAME and proTAME with apcin-M was tested using a Cox proportional hazards model stratified by date, using data from only the experimental blocks in which both proTAME and proTAME with apcin were tested.

Extended Data Figure 9 Model of the effects of apcin and TAME on formation of the APC/C–Cdc20–substrate ternary complex.

a, Schematic drawing of core APC/C subunits. Not all subunits are indicated, and not all known interactions between subunits are illustrated for the sake of simplicity. The light blue oval and polygon indicate binding sites for Cdc20 on core APC/C subunits. b, In the absence of substrate, Cdc20 (green) can bind to the APC/C via the C-box (labelled ‘C’), which interacts with APC8, and the IR-tail (labelled ‘IR’), which interacts with APC3 (Cdc27). c, Binding of substrates (red) that contain a D-box (labelled ‘D’) can promote formation of a co-receptor interaction between the WD-40 domain of Cdc20 and Apc10. d, The RING-containing subunit APC11 can recruit the E2 enzyme to conjugate ubiquitin to the substrate. e, TAME binds APC3 to interfere with the IR-tail binding site. f, Apcin binds to the leucine pocket of the WD-40 domain of Cdc20. g, In the presence of TAME (labelled ‘T’), the IR-binding site is disrupted, but Cdc20 can still be recruited to the APC/C through the C-box interaction and co-receptor interaction. h, Apcin (labelled ‘A’) can disrupt the D-box interaction between the substrate and Cdc20, but Cdc20 can still interact through the C-box and IR-tail interactions. i, Combined use of apcin and TAME disrupts both interactions, cooperatively disrupting the interaction between APC/C, Cdc20 and substrate.

Extended Data Table 1 Data collection and refinement statistics for Cdc20–apcin structure

Supplementary information

Supplementary Information

This file contains Supplementary Methods (synthesis of apcin and apcin-A, statistical methods for analysis of fixed cell and time lapase data), a Supplementary Discussion, and Supplementary References. (PDF 504 kb)

Supplementary Data

R code to generate statistical analysis of combined action of proTAME and apcin in fixed cell imaging assay. (TXT 2 kb)

Supplementary Data

R code to generate statistical analysis of live cell imaging data. (TXT 3 kb)

41586_2014_BFnature13660_MOESM26_ESM.mov

Apcin + proTAME prolongs mitotic duration more than either drug alone in cells treated with control siRNA. RPE1-H2B-GFP cells were treated with 25 μM apcin and/or 6 μM proTAME. The apcin-treated cell divides. The proTAME-treated cell demonstrates abnormal mitotic exit, forming a single mononucleated daughter cell after an attempt at anaphase. The apcin + proTAME –treated cell dies in mitosis. Top row shows Histone H2B-GFP signal, bottom row shows differential interference contrast (DIC). Scale bar indicates 20 μm. (MOV 2881 kb)

Effects of apcin and proTAME in control-siRNA treated cells

Apcin + proTAME prolongs mitotic duration more than either drug alone in cells treated with control siRNA. RPE1-H2B-GFP cells were treated with 25 μM apcin and/or 6 μM proTAME. The apcin-treated cell divides. The proTAME-treated cell demonstrates abnormal mitotic exit, forming a single mononucleated daughter cell after an attempt at anaphase. The apcin + proTAME –treated cell dies in mitosis. Top row shows Histone H2B-GFP signal, bottom row shows differential interference contrast (DIC). Scale bar indicates 20 μm. (MOV 2881 kb)

41586_2014_BFnature13660_MOESM27_ESM.mov

Apcin + proTAME prolongs mitotic duration more than either drug alone in cells treated with Mad2 siRNA. RPE1-H2B-GFP cells were treated with 25 μM apcin and/or 6 μM proTAME. The apcin-treated cell and the proTAME-treated cell divide. The apcin + proTAME –treated cell demonstrates abnormal mitotic exit: the condensed chromatin decondenses, forming a single mononucleated daughter. Another cell in the top left of the same frame dies in mitosis. Top row shows Histone H2B-GFP signal, bottom row shows differential interference contrast (DIC). Scale bar indicates 20 μm. (MOV 2979 kb)

Effects of apcin and proTAME in Mad2-siRNA treated cells

Apcin + proTAME prolongs mitotic duration more than either drug alone in cells treated with Mad2 siRNA. RPE1-H2B-GFP cells were treated with 25 μM apcin and/or 6 μM proTAME. The apcin-treated cell and the proTAME-treated cell divide. The apcin + proTAME –treated cell demonstrates abnormal mitotic exit: the condensed chromatin decondenses, forming a single mononucleated daughter. Another cell in the top left of the same frame dies in mitosis. Top row shows Histone H2B-GFP signal, bottom row shows differential interference contrast (DIC). Scale bar indicates 20 μm. (MOV 2979 kb)

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Sackton, K., Dimova, N., Zeng, X. et al. Synergistic blockade of mitotic exit by two chemical inhibitors of the APC/C. Nature 514, 646–649 (2014). https://doi.org/10.1038/nature13660

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