Characterization of Potent SMAC Mimetics that Sensitize Cancer Cells to TNF Family-Induced Apoptosis.
Members of the Inhibitor of APoptosis (IAP) protein family suppress apoptosis within tumor cells, particularly in the context of immune cell-mediated killing by the tumor necrosis factor (TNF) superfamily cytokines. Most IAPs are opposed endogenously by the second mitochondrial activator of caspases (SMAC), which binds to selected baculovirus IAP repeat (BIR) domains of IAPs to displace interacting proteins. The development of SMAC mimetics as novel anticancer drugs has gained impetus, with several agents now in human clinical trials. To further understand the cellular mechanisms of SMAC mimetics, we focused on IAP family members cIAP1 and cIAP2, which are recruited to TNF receptor complexes where they support cell survival through NF-κB activation while suppressing apoptosis by preventing caspase activation. We established fluorescence polarization (FP) assays for the BIR2 and BIR3 domains of human cIAP1 and cIAP2 using fluorochrome-conjugated SMAC peptides as ligands. A library of SMAC mimetics was profiled using the FP assays to provide a unique structure activity relationship (SAR) analysis compared to previous assessments of binding to XIAP. Potent compounds displayed mean inhibitory binding constants (Ki) of 9 to 27 nM against the BIR3 domains of cIAP1 and cIAP2, respectively. Selected compounds were then characterized using cytotoxicity assays in which a cytokine-resistant human tumor cell line was sensitized to either TNF or lymphotoxin-α (LT-α). Cytotoxicity correlated closely with cIAP1 and cIAP2 BIR3 binding activity with the most potent compounds able to reduce cell viability by 50%. Further testing demonstrated that active compounds also inhibit RIP1 binding to BIR3 of cIAP1 and cIAP2 in vitro and reduce steady-state cIAP1 protein levels in cells. Altogether, these data inform the SAR for our SMAC mimetics with respect to cIAP1 and cIAP2, suggesting that these IAP family members play an important role in tumor cell resistance to cytotoxicity mediated by TNF and LT-α.
Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Rd, La Jolla, CA, 92037, United States of America.
Kate
K
Welsh
Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Rd, La Jolla, CA, 92037, United States of America.
Snezana
S
Milutinovic
Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Rd, La Jolla, CA, 92037, United States of America.
Robert J
RJ
Ardecky
Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Rd, La Jolla, CA, 92037, United States of America.
Marcos
M
Gonzalez-Lopez
Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Rd, La Jolla, CA, 92037, United States of America.
Santhi Reddy
SR
Ganji
Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Rd, La Jolla, CA, 92037, United States of America.
Peter
http://orcid.org/0000-0003-3692-9308
P
Teriete
Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Rd, La Jolla, CA, 92037, United States of America.
Darren
D
Finlay
Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Rd, La Jolla, CA, 92037, United States of America.
Stefan
S
Riedl
Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Rd, La Jolla, CA, 92037, United States of America.
Shu-Ichi
S
Matsuzawa
Torrey Pines Institute for Molecular Studies, 3550 General Atomics Ct, San Diego, CA, 92121, United States of America & 11350 SW Village Parkway, Port St. Lucie, FL, 34987, United States of America.
Clemencia
C
Pinilla
Torrey Pines Institute for Molecular Studies, 3550 General Atomics Ct, San Diego, CA, 92121, United States of America & 11350 SW Village Parkway, Port St. Lucie, FL, 34987, United States of America.
Richard
R
Houghten
Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Rd, La Jolla, CA, 92037, United States of America.
Kristiina
K
Vuori
Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Rd, La Jolla, CA, 92037, United States of America.
John C
JC
Reed
Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Rd, La Jolla, CA, 92037, United States of America.
Nicholas D P
ND
Cosford
D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D045744:Cell Line, Tumor; D005454:Fluorescence Polarization; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D018716:Molecular Mimicry; D011485:Protein Binding; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D051017:Apoptosis Regulatory Proteins; C412403:DIABLO protein, human; D051034:Inhibitor of Apoptosis Proteins; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D014409:Tumor Necrosis Factor-alpha