Blocking XIAP:CASP7-p19 selectively induces apoptosis of CASP3/DR malignancies by a novel reversible small molecule

Abstract

X-linked inhibitor of apoptosis (XIAP) inhibits caspases 3, 7, and 9, thereby preventing cell apoptosis. Endogenous Second mitochondria-derived activator of caspase (Smac) competes out the binding of caspases with XIAP and causes apoptosis, so that Smac mimetics are under clinical trials for anti-cancer chemotherapy. We demonstrated by selectively alkylating caspase 7 (CASP7) to release the active CASP7 for killing the drug-resistant cancer cells with accumulated XIAP:CASP7 resulted from caspase-3 down-regulation (CASP3/DR). However, finding a reversible inhibitor of the protein-protein interaction (PPI) poses a significant challenge. Here, we identified a reversible XIAP:CASP7 inhibitor, 643943, through a multiple-mode virtual screening strategy. In vitro experiments revealed that 643943 bound to CASP7, released the linker-BIR2 domain of XIAP, and activated the caspase. Removing an essential hydroxyl group on 643943 or replacing the OH-interacting Asp93 on CASP7 caused loss of 643943 cytotoxicity, revealing the binding mode. This compound thus selectively killed MCF-7 and other CASP3/DR triple-negative breast cancer cell lines, but not the cancer and normal cell lines expressing higher levels of CASP3 in vitro and in vivo. Moreover, 643943 overcame chemoresistance via down-regulating β-catenin and its associated ABC transporters in paclitaxel-resistant MCF-7 cells. Our studies not only serve as a proof-of-concept for using XIAP:CASP7 as a drug target, but also provide the first reversible XIAP:CASP7 inhibitor for cancer therapy of CASP3/DR malignancies.

Fig. 1. Multiple-mode screening strategy to identify the XIAP:CASP7 PPI inhibitor.

A The cartoon binding model of a small-molecule inhibitor (gray) targeting multiple modes to block the interaction of linker-BIR2 of XIAP (yellow) with CASP7 (blue and light blue) and cause cell apoptosis. B The allosteric binding site, including the exposed Cys246 for I-Lys binding, can be found in both XIAP-bound and XIAP-free form CASP7 structures (PDB codes 1I51 and 1K86). The inhibitor, 643943, binds into modes 1 and 2 to disrupt interactions between CASP7 and XIAP though inducing XIAP-bound to unbound structure and directly stabilizes active-form CASP7. C Multiple-mode strategy increases the chance of 643943 (orange) fitting to the binding site in CASP7. The common site-moiety map was identified by superimposing site-moiety-maps of mode 1 and mode 2 and consists of four core anchors (H1, H2, V1 and V2). D The docked conformation of 643943 at allosteric inhibition site of CASP7. 643943 (orange) interacts with two residues, D93 (anchor H1) and Q243 (anchor H2), through hydrogen bonds (green dashed lines).

Fig. 2. 643943 triggers CASP7-mediated apoptotic signaling by disrupting the XIAP:CASP7 complex.

A Immunoprecipitation/Western blot analysis of XIAP:CASP7 complex in MCF-7 cells and XIAP:CASP3 complex in MCF-10A treated with 643943 (20 μM) for 4 h, respectively. 643943 efficiently blocked the PPI between XIAP and CASP7. GAPDH was used as a loading control. B Cytocidal effects of 643943 on MCF-7 cells based on MTT assay. Cells were treated with 643943 at the indicated concentrations for 24 h and live cells were determined by MTT assay. C Cytocidal effects of 643943 on MCF-7 cells based on sub-G₀ arrest. Cells were treated with 643943 at the indicated concentrations for 24 h. Accumulation of cells in the sub-G₀ fraction was determined by PI-based flow cytometric analysis. D Cytocidal effects of normal MCF-10A cells. Cells were treated with 643943 at the indicated concentrations for 24 h. Accumulation of cells in the sub-G₀ fraction was determined by PI-based flow cytometric analysis. E Determination of intracellular caspase activities in untreated (white bars) or 643943-treated (black bars) MCF-7 cells for 24 h. F Relative apoptosis percentages of MCF-7 cells treated with 20 μM 643943 for 24 h in the absence or presence of the CASP7 inhibitor (MPS) at the indicated doses. G Immunoblotting for CASP7 and GAPDH (control for protein loading) and H relative apoptotic percentages of the MCF-7 cells and the cells transfected with control shRNA (Con-shRNA) or specific shRNA (CASP7-shRNA). A–H Data were obtained from three independent experiments.

Fig. 3. 643943 releases CASP7 from the inactive complex with XIAP.

A In vitro binding assay for GST-XIAP interaction with CASP7 in the presence of increasing 643943 concentrations (0.1 and 0.5 mM). GST-XIAP was pulled down using Glutathione beads and CASP7 was detected via Western blot. Like I-Lys, 643943 disrupted the GST-XIAP:CASP7, but not GST-XIAP:CASP3 complexes. B Activity levels of CASP7 complexed with GST-linker-BIR2 and subsequently treated with 100 μM of different agents including 643943, 119, a peptide AVPFVASLPN derived from SMAC, and a mutant peptide. C Fluorescence spectra of CASP7 in the absence (black) and presence of different concentrations (1, 3, 5, 10, and 15 μM) of 643943 as shown in orange, red, blue, green, and purple, respectively (left panel). The fluorescence spectrum (gray) of 643943 has a λ_max of 420 nm, so that the addition of 643943 did not contribute to the fluorescence changes at 346 nm, the λ_max of CASP7. The right panel shows the changes of fluorescence at 346 nm during titration. D BIAcore traces showing the binding of 0.28 μM GST-linker-BIR2 in the absence (black) and presence of different concentrations of 643943 as shown in orange, yellow, green, cyan, blue, and pink, respectively (left panel) and 119 as a negative control (right panel). The concentrations of 643943 and 119 used were 0, 0.5, 10, 15, 20, 30, and 40 µM. A–D Data were obtained from three independent experiments.

Fig. 4. Allosteric mechanism of 643943 disrupting XIAP:CASP7.

A Sub-G₀ cell populations of MCF-7, MCF-7 transfected with vector only, and MCF-7 variant cells stably expressing CASP7 with D93L mutation (MCF7_D93L) after treatment with I-Lys (1 μM), STS (1 μM), and 643943 (20 μM) for 24 h. B The binding mode of 119, an analog of 643943 without the D93 interacting OH group. C Sub-G₀ cell populations of MCF-7 cells treated with 643943 and 119 measured by flow cytometry. The results showed no 119 induced-sub-G₀ accumulation in MCF-7 cells, suggesting D93 is a key interacting residue for XIAP:CASP7 PPI. D Hydrogen bound interaction networks among significant residues of XIAP:CASP7 interface and four core residues (D93, A96, Q243, and C246) of the allosteric site are shown. D93 is indirectly (via S239) linked to three interacting residues in the linker preceding BIR2, especially D148.

Fig. 5. 643943 suppresses CASP3/DR cancer cells and exhibits anti-tumor activity in vivo.

A, B Western blot analysis and apoptotic cell populations of a panel of breast cancer cell lines treated with 20 μM 643943 for 24 h. The CASP3, CASP7, and XIAP expression levels of these cancer cells were analyzed by Western blot using GADPH as a loading control. Data were obtained from three independent experiments. ** and *** denote p < 0.01 and p < 0.001, respectively. C Body weight of mice. Mice were dosed intraperitoneally with 30% solutol (vehicle, n = 3) or 643943 in 30% solutol at 20 mg/kg (n = 3) once daily for 14 days. Body weights of mice were measured and presented as mean ± SD. D, E Tumor growth of MCF-7 and MDA-MB-468 cells post-treatment without or with 643943. Mice were implanted with MCF-7 (D) or MDA-MB-468 (E) cells and treated without (vehicle, 30% solutol, n = 6) or with 643943 at 20 mg/kg/day (n = 6) for 2 weeks. Tumor volumes were measured every 3 days and presented as mean ± SD. F Analysis of apoptosis, detected by TUNEL. Shown are immunohistochemistry images from representative sections of tumor samples from 643943 and vehicle-treated mice.

Fig. 6. 643943 exhibits synergistic effect with anti-cancer drugs and effectively sensitizes chemoresistant cancer cells to chemotherapy.

A, B Combination treatments with 643943 and anti-cancer drugs (STS and doxorubicin) exhibit synergistic effect in MCF-7 cells. Immunoprecipitation/Western blot analysis of XIAP:CASP7-p19 complex and cleaved PARP1 in MCF-7 cells treated without or with indicated compounds. GAPDH was used as a loading control. C Cell survival analysis following paclitaxel treatment. MCF-7 and paclitaxel-resistant MCF-7 (MCF-7/TR) cells were treated with paclitaxel at the indicated concentrations for 48 h. D G₂M arrest analysis following combination treatment of 643943 and paclitaxel. 643943 treatment significantly recovers the efficacy of paclitaxel in MCF-7/TR. E MTT assays of MCF-7/TR cells treated with 643943, paclitaxel or combination treatment of 643943 and paclitaxel. Cell death of MCF-7/TR cells were analyzed following treatments of 10 nM paclitaxel, 643943 at the indicated concentrations or combination for 24 h. F–H Western blot analysis of β-catenin, ABC transporters, and GAPDH using their specific antibodies in MCF-7/TR treated with DMSO, 5 µM 643943 or 10 µM CASP7 inhibitor (MPS). GAPDH was used as a loading control. A–H Data were obtained from three independent experiments.