The X-linked inhibitor of apoptosis (XIAP) is a promising new molecular target for the design of novel anticancer drugs aiming at overcoming apoptosis-resistance of cancer cells to chemotherapeutic ...agents and radiation therapy. Recent studies demonstrated that the BIR3 domain of XIAP where caspase-9 and Smac proteins bind is an attractive site for designing small-molecule inhibitors of XIAP. Through computational structure-based screening of an in-house traditional herbal medicine three-dimensional structure database of 8221 individual natural products, followed by biochemical testing of selected candidate compounds, we discovered embelin from the Japanese Ardisia herb as a small-molecular weight inhibitor that binds to the XIAP BIR3 domain. We showed that embelin binds to the XIAP BIR3 protein with an affinity similar to that of the natural Smac peptide using a fluorescence polarization-based binding assay. Our NMR analysis further conclusively confirmed that embelin interacts with several crucial residues in the XIAP BIR3 domain with which Smac and caspsase-9 bind. Embelin inhibits cell growth, induces apoptosis, and activates caspase-9 in prostate cancer cells with high levels of XIAP, but has a minimal effect on normal prostate epithelial and fibroblast cells with low levels of XIAP. In stably XIAP-transfected Jurkat cells, embelin effectively overcomes the protective effect of XIAP to apoptosis and enhances the etoposide-induced apoptosis and has a minimal effect in Jurkat cells transfected with vector control. Taken together, our results showed that embelin is a fairly potent, nonpeptidic, cell-permeable, small-molecule inhibitor of XIAP and represents a promising lead compound for designing an entirely new class of anticancer agents that target the BIR3 domain of XIAP.
Persistent expression of certain oncogenes is required for tumor maintenance. This phenotype is referred to as oncogene addiction and has been clinically validated by anticancer therapies that ...specifically inhibit oncoproteins such as BCR-ABL, c-Kit, HER2, PDGFR, and EGFR. Identifying additional genes that are required for tumor maintenance may lead to new targets for anticancer drugs. Although the role of aberrant Wnt pathway activation in the initiation of colorectal cancer has been clearly established, it remains unclear whether sustained Wnt pathway activation is required for colorectal tumor maintenance. To address this question, we used inducible β-catenin shRNAs to temporally control Wnt pathway activation in vivo. Here, we show that active Wnt/β-catenin signaling is required for maintenance of colorectal tumor xenografts harboring APC mutations. Reduced tumor growth upon β-catenin inhibition was due to cell cycle arrest and differentiation. Upon reactivation of the Wnt/β-catenin pathway colorectal cancer cells resumed proliferation and reacquired a crypt progenitor phenotype. In human colonic adenocarcinomas, high levels of nuclear β-catenin correlated with crypt progenitor but not differentiation markers, suggesting that the Wnt/β-catenin pathway may also control colorectal tumor cell fate during the maintenance phase of tumors in patients. These results support efforts to treat human colorectal cancer by pharmacological inhibition of the Wnt/β-catenin pathway.
The blockade of aberrant hedgehog (Hh) signaling has shown promise for therapeutic intervention in cancer. A cell-based phenotypic high-throughput screen was performed, and the lead structure (1) was ...identified as an inhibitor of the Hh pathway via antagonism of the Smoothened receptor (Smo). Structure−activity relationship studies led to the discovery of a potent and specific Smoothened antagonist N-(6-((2S,6R)-2,6-dimethylmorpholino)pyridin-3-yl)-2-methyl-4′-(trifluoromethoxy)biphenyl-3-carboxamide (5m, NVP-LDE225), which is currently in clinical development.
Abnormal activation of the Hedgehog (Hh) signaling pathway has been linked to several types of human cancers, and the development of small-molecule inhibitors of this pathway represents a promising ...route toward novel anticancer therapeutics. A cell-based screen performed in our laboratories identified a new class of Hh pathway inhibitors, 1-amino-4-benzylphthalazines, that act via antagonism of the Smoothened receptor. A variety of analogues were synthesized and their structure−activity relationships determined. This optimization resulted in the discovery of high affinity Smoothened antagonists, one of which was further profiled in vivo. This compound displayed a good pharmacokinetic profile and also afforded tumor regression in a genetic mouse model of medulloblastoma.
Bcl-2 belongs to a growing family of proteins which regulates programmed cell death (apoptosis). Overexpression of Bcl-2 has been observed in 70% of breast cancer, 30−60% of prostate cancer, 80% of ...B-cell lymphomas, 90% of colorectal adenocarcinomas, and many other forms of cancer. Thereby, Bcl-2 is an attractive new anti-cancer target. Herein, we describe the discovery of novel classes of small-molecule inhibitors targeted at the BH3 binding pocket in Bcl-2. The three-dimensional (3D) structure of Bcl-2 has been modeled on the basis of a high-resolution NMR solution structure of Bcl-XL, which shares a high sequence homology with Bcl-2. A structure-based computer screening approach has been employed to search the National Cancer Institute 3D database of 206 876 organic compounds to identify potential Bcl-2 small-molecule inhibitors that bind to the BH3 binding site of Bcl-2. These potential Bcl-2 small-molecule inhibitors were first tested in an in vitro binding assay for their potency in inhibition of the binding of a Bak BH3 peptide to Bcl-2. Thirty-five potential inhibitors were tested in this binding assay, and seven of them were found to have a binding affinity (IC50 value) from 1.6 to 14.0 μM. The anti-proliferative activity of these seven active compounds has been tested using a human myeloid leukemia cell line, HL-60, which expresses the highest level of Bcl-2 protein among all the cancer cell lines examined. Compound 6 was the most potent compound and had an IC50 value of 4 μM in inhibition of cell growth using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Five other compounds had moderate activity in inhibition of cell growth. Compound 6 was further evaluated for its ability to induce apoptosis in cancer cells. It was found that 6 induces apoptosis in cancer cells with high Bcl-2 expression and its potency correlates with the Bcl-2 expression level in cancer cells. Furthermore, using NMR methods, we conclusively demonstrated that 6 binds to the BH3 binding site in Bcl-XL. Our results showed that small-molecule inhibitors of Bcl-2 such as 6 modulate the biological function of Bcl-2, and induce apoptosis in cancer cells with high Bcl-2 expression, while they have little effect on cancer cells with low or undetectable levels of Bcl-2 expression. Therefore, compound 6 can be used as a valuable pharmacological tool to elucidate the function of Bcl-2 and also serves as a novel lead compound for further design and optimization. Our results suggest that the structure-based computer screening strategy employed in the study is effective for identifying novel, structurally diverse, nonpeptide small-molecule inhibitors that target the BH3 binding site of Bcl-2.
Gossypol, a male contraceptive drug, has been demonstrated to have antiproliferative and antimetastatic effects on many kinds of cancer cells
in vitro. HT-29 human carcinoma cell line is one of the ...most susceptible cell lines to gossypol-induced cell death. Here, it is shown that treatment of HT-29 cells with gossypol not only induces cell cycle arrest on the G0/G1 phase, but also induces apoptosis. With a serial of Western blot analysis, it is revealed that gossypol-induced cell cycle arrest is involved in P21 up-regulation and cyclin D1 down-regulation; gossypol-induced apoptosis triggers down-regulation of anti-apoptosis Bcl-2 members: Bcl-X
L, Bag-1 and Mcl-1, up-regulation of pro-apoptosis Bcl-2 member Bak, activation of caspase-3, -6, -7, -8, and -9, up-regulation of Apaf-1, release of cytochrome
c (cyto-
c) from mitochondria, and activation of both DFF45 and PARP. Taken together, gossypol-induced cell death initiates extensive alterations of cell cycle and apoptosis proteins. Gossypol-induced apoptosis of HT-29 cells is through first the mitochondrial pathway, then the death receptor pathway, and the mitochondria pathway is, at least in part, involved in cyto-
c release.
LIGHT is a new member of the tumor necrosis factor superfamily, which binds to lymphotoxin β receptor, herpes virus entry mediator, or TR6. This work was carried out to elucidate the molecular ...mechanism of LIGHT-sensitized, interferon gamma (IFNγ)-mediated apoptosis of MDA-MB-231 cells. It was revealed that LIGHT treatment resulted in down-regulation of anti-apoptosis Bcl-2 family member: Bcl-2, Bcl-X
L, Bag-1, and Mcl-1; up-regulation of pro-apoptosis Bcl-2 family member: Bak and Ser (112)-phosphor-Bad; down-regulation of pro-apoptosis Bcl-2 member Bax; the other pro-apoptosis member Bid remains unaltered. LIGHT treatment also resulted in activation of caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, DFF45, and PARP. However, caspase activation and caspase activity, especially caspase-3 activity, is not required for LIGHT-induced apoptosis of MDA-MB-231 cells, since caspase-3 inhibitor, benzyloxycarbonyl–Asp–Glu–Val–Asp-fluoromethylketone, and a broad range caspase inhibitor, benzyloxycarbonyl–val–ala–asp–fluoromethylketone failed to block the apoptosis induced by LIGHT and IFNγ in MDA-MB-231 cells. In summary, LIGHT-sensitized IFNγ-mediated apoptosis of MDA-MB-231 cells is probably through down-regulation of anti-apoptosis Bcl-2 family members; it could be caspase (especially caspase-3)-independent, even though extensive caspase activation was observed.
Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in detail novel properties of the type II RAF inhibitor, LXH254.
LXH254 was profiled in biochemical,
, and
...assays, including examining the activities of the drug in a large panel of cancer-derived cell lines and a comprehensive set of
models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF.
We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and
mutants, but had only modest activity in
mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally,
, LXH254 drove complete regressions of isogenic variants of RAS-mutant cells lacking ARAF expression, while parental lines were only modestly sensitive.
LXH254 is a novel RAF inhibitor, which is able to inhibit dimerized BRAF and CRAF, as well as monomeric BRAF, while largely sparing ARAF.
While most SH2 domains bind phosphotyrosyl (pTyr) containing peptides in extended fashion, the growth factor receptor-bound protein 2 (Grb2) SH2 domain preferentially binds ligands in bend ...conformations. Accordingly, incorporation of bend-inducing functionality into synthetic ligands could potentially enhance their affinity for this SH2 domain. A macrocyclic tripeptide mimetic that contains a simplified pTyr surrogate lacking an α-nitrogen has recently been shown to exhibit high Grb2 SH2 domain-binding affinity in extracellular ELISA-based assays. However, the same compound is largely ineffective in whole-cell assays. It is known that acidic functionality originating from the α-nitrogen of pTyr residues or from the α-position of P0 pTyr mimetics not only increases binding affinity of peptides to Grb2 SH2 domains in extracellular assays but also enhances potency in cell-based systems. Such functionality is absent from the previously reported macrocycle. Therefore, the current study was undertaken to examine the effects of introducing carboxylic functionality at the pTyr mimetic α-position of macrocyclic ligands. It was found that such a modification not only enhanced Grb2 SH2 domain binding in extracellular assays but also conferred high efficacy in whole-cell systems. The most potent compound of the current study exhibited an IC50 value of 0.002 μM in an extracellular ELISA-based assay, and in MDA-MB-453 cells, it both inhibited the association of Grb2 with p185erbB-2 and exhibited antimitogenic effects with submicromolar IC50 values.
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