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

Simultaneous disruption of two different protein–protein interactions within the (APC/C–Cdc20)–substrate complex can synergistically inhibit APC/C-dependent proteolysis and mitotic exit. The anaphase-promoting complex/cyclosome (APC/C) is a thirteen-subunit ubiquitin ligase enzyme that mediates progression through mitotic cell division by targeting substrate proteins for degradation by the ubiquitin–proteasome system. During mitosis, the protein Cdc20 binds to APC/C to activate it, and the two proteins together recognize target substrates. Blocking mitotic exit by blocking APC/C activity could induce tumour-cell death and therefore might be of potential therapeutic value. This study shows that simultaneous disruption of two different protein–protein interactions within the (APC/C–Cdc20)-substrate complex can synergistically inhibit APC/C-dependent proteolysis and mitotic exit. 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.