title: 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 proteasome. Because blocking mitotic exit is an effective approach for inducing tumour cell death, 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). 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 interaction. This work suggests that simultaneous disruption of multiple, weak protein-protein interactions is an effective approach for inactivating a protein machine.
authors.affiliation: 1] Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA [2].
authors.forename: Katharine L
authors.initials: KL
authors.lastname: Sackton
authors.affiliation: 1] Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA [2].
authors.forename: Nevena
authors.initials: N
authors.lastname: Dimova
authors.affiliation: 1] Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA [2].
authors.forename: Xing
authors.initials: X
authors.lastname: Zeng
authors.affiliation: 1] Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, Texas 75390, USA [2] Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China (W.T.); Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA (K.L.P.); Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (F.S.). [3].
authors.forename: Wei
authors.initials: W
authors.lastname: Tian
authors.affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
authors.forename: Mengmeng
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford Street, Cambridge, Massachusetts 02138, USA.
authors.forename: Timothy B
authors.initials: TB
authors.lastname: Sackton
authors.affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
authors.forename: Johnathan
authors.initials: J
authors.lastname: Meaders
authors.affiliation: 1] Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA [2] Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China (W.T.); Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA (K.L.P.); Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (F.S.).
authors.forename: Kathleen L
authors.initials: KL
authors.lastname: Pfaff
authors.affiliation: 1] Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA [2] Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China (W.T.); Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA (K.L.P.); Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (F.S.).
authors.forename: Frederic
authors.initials: F
authors.lastname: Sigoillot
authors.affiliation: 1] Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, Texas 75390, USA [2] Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, Maryland 20815, USA.
authors.forename: Hongtao
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, Texas 75390, USA.
authors.forename: Xuelian
authors.initials: X
authors.lastname: Luo
authors.affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
authors.forename: Randall W
authors.initials: RW
authors.lastname: King
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D002219:Carbamates / Q000494:pharmacology*; D064199:Cdc20 Proteins / Q000737:chemistry / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D018360:Crystallography, X-Ray; D003959:Diamines / Q000494:pharmacology*; D004357:Drug Synergism; D008938:Mitosis / Q000187:drug effects*; D011485:Protein Binding / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D014106:Tosylarginine Methyl Ester / Q000494:pharmacology*; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D002219:Carbamates; D064199:Cdc20 Proteins; D003959:Diamines; C000593255:apcin; D014106:Tosylarginine Methyl Ester; D064173:Anaphase-Promoting Complex-Cyclosome