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. 2021 Nov 26:19:6366-6374.
doi: 10.1016/j.csbj.2021.11.034. eCollection 2021.

Structure-based identification of a new IAP-targeting compound that induces cancer cell death inducing NF-κB pathway

Federica Cossu  1 , Simone Camelliti  1   2 , Daniele Lecis  2 , Luca Sorrentino  1   3 , Maria Teresa Majorini  2 , Mario Milani  1 , Eloise Mastrangelo  1
Affiliations

Structure-based identification of a new IAP-targeting compound that induces cancer cell death inducing NF-κB pathway

Federica Cossu et al. Comput Struct Biotechnol J. .

Abstract

Inhibitors of apoptosis proteins (IAPs) are validated onco-targets, as their overexpression correlates with cancer onset, progression, diffusion and chemoresistance. IAPs regulate cell death survival pathways, inflammation, and immunity. Targeting IAPs, by impairing their protein-protein interaction surfaces, can affect events occurring at different stages of cancer development. To this purpose, we employed a rational virtual screening approach to identify compounds predicted to interfere with the assembly of pro-survival macromolecular complexes. One of the candidates, FC2, was shown to bind in vitro the BIR1 domains of both XIAP and cIAP2. Moreover, we demonstrated that FC2 can induce cancer cell death as a single agent and, more potently, in combination with the Smac-mimetic SM83 or with the cytokine TNF. FC2 determined a prolonged activation of the NF-κB pathway, accompanied to a stabilization of XIAP-TAB1 complex. This candidate molecule represents a valuable lead compound for the development of a new class of IAP-antagonists for cancer treatment.

Keywords: BIR, baculoviral IAP repeat; Baculoviral IAP repeat; Drug discovery; IAP, inhibitor of apoptosis proteins; IAP-antagonist; Inhibitor of apoptosis proteins; NF-kB; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; SM, Smac-mimetic; TAB, TAK1 binding protein; TAK1, (TGFβ)-activated kinase; TGFβ, transforming growth factor-β; TNF, tumor necrosis factor; TRAF, TNF receptor associated factor; Virtual screening.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

None
Graphical abstract
Fig. 1
Fig. 1
Comparison between FC2 poses in virtual docking versus cIAP1-, cIAP2- and XIAP-BIR1 α1-α2 structural hotspots. A) The BIR1 domain of homologous IAPs shown as grey surface. The structural hotspots involved in PPI with the partner proteins (TRAF2 or TAB1), the α1-α2 region of BIR1, are shown as red surface. FC2 (lines) predicted poses on cIAP1- (B, orange cartoon), cIAP2- (C, light blue cartoon) and XIAP-BIR1 (D, green cartoon) were superimposable, showing comparable free energies of binding (ΔG for cIAP2-BIR1/XIAP-BIR1 = −8.2 kcal/mol; ΔG for cIAP1-BIR1 = −8.7 kcal/mol). In sticks are highlighted the residues of α1-α2 region involved in the predicted interaction with FC2. Images were drawn with PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
SAR study with FC2 analogs in cell viability assays. A) Chemical structure of FC2. B) Chemical structure of nine FC2 analogs available in the database (https://www.hit2lead.com/). The FC2-analogs have different chemical substitutions in R1, R2 and R3. C) Viability of MDA-MB-231 cells treated with FC2 and FC2-analogs at 3 different concentrations (1, 10, 100 μM). Among the FC2-analog compounds tested, FC2 shows the highest cytotoxicity at intermediate concentrations (10 μM, light grey bar). Significant cytotoxicity is observed for analog 4 at the highest concentration (100 μM, black bar). Mean values ± Standard deviation are shown.
Fig. 3
Fig. 3
FC2 activity as a single agent and in combination with TNF. (A) Viability of MDA-MB-231 cells treated with FC2 alone (white bars) and in combination with recombinant TNF (50 ng/ml; black bars). (B) TNF-independent cytotoxicity of the FC2: MDA-MB-231 cell treated with different amounts of FC2 (from 0 to 100 μM) in the absence (red line) / presence (black line) of IFX (which blocks autocrine TNF). All the experiments were repeated both in technical (three for each experiment) and biological (three independent experiments) triplicate. Mean values ± Standard deviation are shown. Statistical analysis performed with GraphPad. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
Enhanced FC2 cytotoxic activity in presence of SM. Effect on cell viability of the combination of FC2 and SM83: (A) MDA-MB-231 and (B) SK-OV3 cells pretreated with different concentration of SM83, followed by addition of FC2 [0 (white bars), 12.5 (grey bars) and 100 μM (black bars)]. The experiments were repeated both in technical (three for each experiment) and biological (three independent experiments) triplicate. Mean values ± Standard deviation are shown.
Fig. 5
Fig. 5
Effects of FC2 on the NF-κB pathway in MDA-MB-231 and HEK293 cells. A) MDA-MB-231 cells treated with 20 μM FC2 and then stimulated with 50 ng/mL TNF. NF-κB markers (pp65, pTAK1, cIAP1, cIAP2, XIAP, pMKK3/6, MKK6 and IκBα) were chosen to monitor the activation of the pathway. (B) In the presence of FC2, pp65 levels increase, suggesting a more prominent phosphorylation of p65 and NF- κB activation, and this effect is particularly evident at 1 and 5 h. Along these time ranges also IκBα levels are significantly different in the absence/presence of FC2, indicating a delay in protein levels’ recovery upon TNF-induced degradation. Densitometry analysis was performed with ImageQuant 5.2. C) Immunoprecipitation of XIAP overexpressed in HEK293. Left panel: lysates; right panel, immunoprecipitation of XIAP with Myc revealed a more pronounced recruitment of TAB1 and TAK1 upon treatment with FC2.
Fig. 6
Fig. 6
FC2 increases p65 translocation to the nucleus. A) MDA-MB-231 cells treated with 100 μM FC2 and then stimulated with 50 ng/mL TNF. Extraction of cytoplasmic and nuclear proteins revealed the decrease of p65 levels in the cytoplasm and an increase of p65 levels in the nucleus, suggesting a more pronounced activation of NF-κB pathway upon treatment with FC2. B) The effect of FC2 on p65 translocation to the nucleus is appreciable in the densitometry graph (ImageQuant 5.2), where the signal of p65 was normalized versus HDAC1.
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