Cellular, biochemical, and genetic analysis of mechanism of small molecule IAP inhibitors.
XIAP is member of the IAP family of anti-apoptotic proteins and is known for its ability to bind and suppress caspase family cell death proteases. A phenylurea series of chemical inhibitors of XIAP was recently generated by our laboratories (Schimmer, A. D., Welsh, K., Pinilla, C., Bonneau, M., Wang, Z., Pedersen, I. M., Scott, F. L., Glinsky, G. V., Scudiero, D. A., Sausville, E., Salvesen, G., Nefzi, A., Ostresh, J. M., Houghten, R. A., and Reed, J. C. (2004) Cancer Cell 5, 25-35). We examined the mechanisms of action of these chemical compounds using biochemical, molecular biological, and genetic methods. Active phenylurea-based compounds dissociated effector protease caspase-3 but not initiator protease caspase-9 from XIAP in vitro and restored caspase-3 but not caspase-9 enzymatic activity. When applied to tumor cell lines in culture, active phenylurea-based compounds induced apoptosis in a rapid, concentration-dependent manner, associated with activation of cellular caspases. Apoptosis induced by active phenylurea-based compounds was blocked by chemical inhibitors of caspases, with inhibitors of downstream effector caspases displaying more effective suppression than inhibitors of upstream initiator caspases. Phenylurea-based XIAP antagonists induced apoptosis (defined by annexin V staining) prior to mitochondrial membrane depolarization, in contrast to cytotoxic anticancer drugs. Consistent with these findings, apoptosis induced by phenylurea-based compounds was not altered by genetic alterations in the expression of Bcl-2 family proteins that control mitochondria-dependent cell death pathways, including over-expression of anti-apoptotic proteins Bcl-2 or Bcl-X(L) and genetic ablation of pro-apoptotic proteins Bax and Bak. Conversely, conditional over-expression of an active fragment of XIAP or genetic ablation of XIAP expression altered the apoptosis dose-response of the compounds. Altogether, these findings indicate that phenylurea-based XIAP antagonists block interaction of downstream effector caspases with XIAP, thus inducing apoptosis of tumor cell lines through a caspase-dependent, Bcl-2/Bax-independent mechanism.
Wang
Zhiliang
Z
The Burnham Institute, La Jolla, California 92037, USA.
Cuddy
Michael
M
Samuel
Temesgen
T
Welsh
Kate
K
Schimmer
Aaron
A
Hanaii
Farid
F
Houghten
Richard
R
Pinilla
Clemencia
C
Reed
John C
JC
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006801:Humans; D007192:Indenes / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D010671:Phenylurea Compounds / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015843:Serpins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism; D051636:X-Linked Inhibitor of Apoptosis Protein; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C494772:BAK1 protein, human; C494826:BAX protein, human; C494811:BCL2L1 protein, human; C494773:Bak1 protein, mouse; C494827:Bax protein, mouse; C494812:Bcl2l1 protein, mouse; D015853:Cysteine Proteinase Inhibitors; D007192:Indenes; D008565:Membrane Proteins; D010671:Phenylurea Compounds; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D015843:Serpins; D014764:Viral Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; C079101:1-(7-(2-hydroxyethyl)dodecahydro-3a-methyl-1H-benz(e)inden-3-yl)ethanone; C074641:interleukin-1beta-converting enzyme inhibitor; C505407:CASP3 protein, human; C506433:CASP9 protein, human; C505409:Casp3 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases