Fig 1.
Effect of buffer on KD of SMAC peptide binding to BIR domains.
All assays contained TCEP at 1 mM, 0.005% Tween 20 and SMAC-rhodamine at 20 nM. The buffers used were PBS @ pH 7.4, 25 mM HEPES @ pH 7.5, 25 mM HEPES @ pH 7.5 with 20 mM β-glycerol phosphate, 10 mM Potassium Phosphate @ pH 7.4, or 50 mM TRIS @ pH 7.5. Proteins were diluted into 25 mM HEPES @ pH 7.5 with 1 mM TCEP. FPA data were collected on the Analyst at 0, 30 and 60 min. Time overlays are plotted in the figure. KDs were determined in Prism.
Fig 2.
KD determination of SMAC-rhodamine binding to BIR2 and BIR3 domains of cIAP1 and cIAP2.
Data were for assay conditions consisting of 25 mM Hepes @ 7.5, 1 mM TCEP, 20 nM SMAC-rhodamine with varying concentrations of various BIR domains. For cIAP1-BIR3, assays included 40 mM β-glycerol phosphate. Plates were read on the Analyst and observed mP were plotted against the log of protein concentration.
Fig 3.
Competition of SMAC-7-mer with SMAC-rhodamine.
Assays conditions were 25 mM Hepes @ pH 7.5, 1 mM TCEP, 0.005% Tween 20 and 20 nM SMAC-rhodamine. Where cIAP1-BIR3 was present, 40 mM β-glycerol phosphate was also present in the assay. Proteins were present at ~50 nM for cIAP1-BIR3, 125 nM for cIAP2-BIR3, and at 1 μM for both cIAP1-BIR2 and cIAP2-BIR2. SMAC peptide (AVPIAQK) ranged between ~6 nM and 100 μM.
Table 1.
Binding affinities of tetrahydronaphthyl series SMAC mimetic compounds for BIR domains of cIAP1 and cIAP2a.
Table 2.
Binding affinities of naphthyl series SMAC mimetic compounds for BIR domains of cIAP1 and cIAP2a.
Table 3.
Binding affinities of hydrazine series SMAC mimetic compounds for BIR domains of cIAP1 and cIAP2a.
Fig 4.
Condensed structures (A,B, and C) of the compounds series described in Tables 1, 2 and 3. (D) Structure of P2 substituent of compound 18.
Table 4.
EC50 Determinations of selected and reference compounds with LT-α and TNF.
Fig 5.
SMAC analogues sensitize PC-3 tumor cells to TNF and LT-α.
Representative curves are shown in this figure for potent, moderate, weak and inactive analogues. Five thousand cells were seeded per well of 96 well plates and incubated overnight in culture medium. The following day, compounds were added and then 10 nM TNF or 90 pM LT-α was administered to the experimental wells, while media was added to control wells. Incubation was continued for 24 h. and then CellTiter-Glo was added to each well and the plates were read in a luminometer. EC50 values were determined with a nonlinear fit using Prism software. The most potent compounds were tested at a lower concentration range (see 100 nM point highlighted by box).
Fig 6.
Correlation between Ki of the cIAP BIR domains and their EC50.
Log of the Ki values for SMAC peptide displacement as measured by FPA was plotted against cell viability EC50 values using the data shown in Fig 5 for either TNF or LT-α. Correlations coefficient (r) and p-values are indicated.
Fig 7.
SMAC mimetic 38 induces degradation of cIAP1.
MDA-MB-231 cells were seeded at 40,000 per well of 12 well plates and cultured overnight. The next day, cultures were either left untreated (No trtm) or were treated for 6 h. with DMSO, 5 μM of SMAC mimetic 38 or 5 μM of inactive analogue compound 40. Cells were lysed in SDS-sample buffer and lysates were analyzed by SDS-PAGE/immunoblotting using antibodies specific for cIAP1 and beta-actin. Molecular weight markers are indicated in kilo-Daltons (kDa).
Fig 8.
SMAC mimetics inhibit cIAP1 and cIAP2 binding to RIP1.
HEK293T cells were transfected with myc-RIP1 plasmid and 24h later, cell lysates were prepared and divided into equal aliquots to which 7 μg of either GST-cIAP1-BIR3 or GST-cIAP2-BIR3 was added. Then, aliquots of either DMSO control or various concentrations of SMAC mimetics 37, 38 or inactive analogue 40 were added. After overnight incubation, GST-cIAP1-BIR3 or GST-cIAP2-BIR3 proteins were recovered using glutathione-sepharose beads and the bound myc-RIP1 protein was detected by SDS-PAGE/immunoblotting using anti-myc antibody for detection of myc-RIP1 and anti-GST antibody for detection of GST-fusion proteins.
Fig 9.
Structures of compounds described in Table 4.