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

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

Expedient Synthesis of Highly Potent Antagonists of Inhibitor of Apoptosis Proteins (IAPs) with Unique Selectivity for ML-IAP

A series of novel, potent antagonists of the inhibitor of apoptosis proteins (IAPs) were synthesized in a highly convergent and rapid fashion (≤6 steps) using the Ugi four-component reaction as the key step, thus enabling rapid optimization of binding potency. These IAP antagonists compete with caspases 3, 7, and 9 for inhibition by X chromosome-linked IAP (XIAP) and bind strongly (nanomolar binding constants) to several crucial members of the IAP family of cancer pro-survival proteins to promote apoptosis, with a particularly unique selectivity for melanoma IAP (ML-IAP). Experiments in cell culture revealed powerful cancer cell growth inhibitory activity in multiple (breast, ovarian, and prostate) cell lines with single agent toxicity at low nanomolar levels against SKOV-3 human ovarian carcinoma cells. Administration of the compounds to human foreskin fibroblast cells revealed no general toxicity to normal cells. Furthermore, computational modeling was performed, revealing key contacts between the IAP proteins and antagonists, suggesting a structural basis for the observed potency

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

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

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

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

Structures of compounds described in Table 4.

<p>Structures of compounds described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#pone.0161952.t004" target="_blank">Table 4</a>.</p