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.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Protein Data Bank
Structure coordinates have been deposited in Protein Data Bank under accession number 4N14.
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.
Extended data figures
Extended data tables
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.
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.
About this article
Nature Chemical Biology (2017)