Characterization of Potent SMAC Mimetics that Sensitize Cancer Cells to TNF Family-Induced Apoptosis - Fig 4

<p>Condensed structures (A,B, and C) of the compounds series described in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#pone.0161952.t001" target="_blank">1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#pone.0161952.t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#pone.0161952.t003" target="_blank">3</a>. (D) Structure of P₂ substituent of compound <b>18</b>.</p

Binding affinities of naphthyl series SMAC mimetic compounds for BIR domains of cIAP1 and cIAP2^a.

<p>Binding affinities of naphthyl series SMAC mimetic compounds for BIR domains of cIAP1 and cIAP2<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#t002fn002" target="_blank">^a</a>.</p

SMAC mimetic 38 induces degradation of cIAP1.

<p>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 <b>38</b> or 5 μM of inactive analogue compound <b>40</b>. 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).</p

Correlation between K_i of the cIAP BIR domains and their EC₅₀.

<p>Log of the K_i values for SMAC peptide displacement as measured by FPA was plotted against cell viability EC₅₀ values using the data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#pone.0161952.g005" target="_blank">Fig 5</a> for either TNF or LT-α. Correlations coefficient (r) and p-values are indicated.</p

SMAC mimetics inhibit cIAP1 and cIAP2 binding to RIP1.

<p>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 <b>37</b>, <b>38</b> or inactive analogue <b>40</b> 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.</p

Effect of buffer on K_D of SMAC peptide binding to BIR domains.

<p>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. K_Ds were determined in Prism.</p

K_D determination of SMAC-rhodamine binding to BIR2 and BIR3 domains of cIAP1 and cIAP2.

<p>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.</p

Binding affinities of hydrazine series SMAC mimetic compounds for BIR domains of cIAP1 and cIAP2^a.

<p>Binding affinities of hydrazine series SMAC mimetic compounds for BIR domains of cIAP1 and cIAP2<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#t003fn002" target="_blank">^a</a>.</p

Competition of SMAC-7-mer with SMAC-rhodamine.

<p>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.</p

EC₅₀ Determinations of selected and reference compounds with LT-α and TNF.

<p>EC₅₀ Determinations of selected and reference compounds with LT-α and TNF.</p