研究人员用所谓的“碳化氢钉针(hydrocarbon stapling)”技术将一个起稳定作用的小分子化合物植入一个试验性癌症药物，提高了该药物促进白血病细胞自杀的效果。这项新的研究发现，用小分子稳定住的肽药物降低了移植到小鼠上的人类肿瘤的增长。一篇相关的研究评述指出，将非天然氨基酸准确地添加到用全天然氨基酸制造的具有生物活性的肽中，能帮助避免这类肽药物的一些常见弊端，包括药效低、不稳定、以及将药物送到细胞的效率低等。
在另一项有关的研究中，Lin Li和同事合成了一个小分子，它能模仿蛋白质-蛋白质的相互作用，来诱导肿瘤细胞选择性的凋亡。这个化合物在治疗发炎疾病上也具有潜力。人们认为，了解凋亡调节的细节可能对发现更有效的癌症药物有用，这项研究为此提供了一些希望。
Activation of Apoptosis in Vivo by a Hydrocarbon-Stapled BH3 Helix
BCL-2 family proteins constitute a critical control point for the regulation of apoptosis. Protein interaction between BCL-2 members is a prominent mechanism of control and is mediated through the amphipathic -helical BH3 segment, an essential death domain. We used a chemical strategy, termed hydrocarbon stapling, to generate BH3 peptides with improved pharmacologic properties. The stapled peptides, called "stabilized alpha-helix of BCL-2 domains" (SAHBs), proved to be helical, protease-resistant, and cell-permeable molecules that bound with increased affinity to multidomain BCL-2 member pockets. A SAHB of the BH3 domain from the BID protein specifically activated the apoptotic pathway to kill leukemia cells. In addition, SAHB effectively inhibited the growth of human leukemia xenografts in vivo. Hydrocarbon stapling of native peptides may provide a useful strategy for experimental and therapeutic modulation of protein-protein interactions in many signaling pathways.
Fig. 1. Enhanced helicity, protease resistance, and serum stability of hydrocarbon-stapled BID BH3 compounds. (A and B) ,-disubstituted nonnatural amino acids containing olefinic side chains of varying length were synthesized as previously reported (16, 31, 32). Nonnatural amino acid substitutions were made to flank three (substitution positions i and i+4) or six (i and i+7) amino acids within the BID BH3 peptide, so that reactive olefinic residues would reside on the same face of the helix. (C) Circular dichroism was used to measure the percentages of SAHB maintained in helical configuration when dissolved in aqueous potassium phosphate solution (pH7) (supporting online material). (D) Fluoresceinated SAHBA and BID BH3 peptide were incubated at 37°C in mouse serum or injected intravenously (10 mg/kg) into NOD SCID mice. Serum concentrations of SAHBA and BID BH3 peptide were measured at the indicated time points with a fluorescence-based high-performance liquid chromatography detection assay. Both assays demonstrated enhanced serum stability of SAHBA.
Fig. 2. SAHBA targets the binding pocket of BCL-XL, displays enhanced BCL-2 binding affinity, and specifically activates cytochrome c release from mitochondria in vitro. (A) HSQC experiments show similar spectral changes in 15N-BCL-XL upon binding SAHBA or BID BH3 peptide. (B) Kd's for binding of individual peptides to glutathione S-transferase–BCL-2 were determined by fluorescence polarization. (C) Mouse liver mitochondria (wild-type or Bak–/–, 0.5 mg/ml) were incubated for 40 min with 25 to 200 nM concentrations of BID BH3 peptide, SAHBA, or SAHBA(GE), and cytochrome c was measured in the supernatant and sedimented mitochondria by an enzyme-linked immunosorbent assay.
Fig. 3. SAHBA penetrates Jurkat leukemia cells by fluid-phase endocytosis and localizes to the mitochondrial membrane. Jurkat leukemia cells were incubated with FITC-labeled peptides for 4 hours at 37°C, followed by FACS analysis (A). FITC-SAHBA uptake occurred in a time-dependent manner at 37°C (B), but no FITC-SAHBA labeling was evident by 4 hours, when the experiment was performed at 4°C (C). Live confocal images demonstrated a colocalization of FITC-SAHBA with 4.4-kD dextran-labeled endosomes (D) but not transferrin-labeled endosomes (E) at 4 hours. A mitochondrial colocalization was evident by 24 hours, as demonstrated by the merged images of FITC-SAHBA and MitoTracker in live cells (F) and those of FITC-SAHBA and Tom20 (a mitochondrial outer-membrane marker) in fixed cells (G). Arrows highlight sites of colocalization corresponding to the surface of mitochondria cut in cross section (G).
Fig. 4. SAHBA triggers apoptosis in Jurkat cells and inhibits a panel of human leukemia cells. FACS analysis of annexin V–treated cells was used to monitor apoptosis of Jurkat cells treated with 0.5 to 5 µM concentrations of BID BH3 peptide, SAHBA, or SAHBA(GE) for 20 hours (A). Jurkat, REH, MV4;11, SEMK2, and RS4;11 leukemia cells were treated with serial dilutions of SAHBA (B), BID BH3 peptide (C), or SAHBA(GE) (D), and MTT assays were performed at 48 hours to measure viability.
Fig. 5. SAHBA suppresses growth of human leukemia cells in vivo, prolonging the survival of leukemic mice. (A) Leukemic SCID beige mice [with a day-1 natural logarithm (ln) bioluminescence range of 14.4 to 15.9] were treated with intravenous injections of 10 mg/kg SAHBA or vehicle (5% DMSO in D5W) daily for 7 days and were monitored for survival; leukemia burden was quantified by total body luminescence (photons/s/mouse) on days 1, 3, and 5. The disease course from days 3 to 5 differed between SAHBA-treated animals and controls (P = 0.016, Fisher's exact test [box in (A)], as illustrated by representative Xenogen images of bioluminescent leukemic mice (B); red signal represents the highest level of leukemia on the colorimetric scale. (C) Median survival was prolonged in SAHBA-treated animals as compared to controls (P = 0.004, log rank test). (D) To compare SAHBA with SAHBA(GE), leukemic mice (with a day 1 ln bioluminescence range of 17.1 to 17.9) were treated daily with SAHB (10 mg/kg) or vehicle, and animals were imaged on days 1 and 3 to measure total body luminescence.
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A Small Molecule Smac Mimic Potentiates TRAIL- and TNF-Mediated Cell Death
We describe the synthesis and properties of a small molecule mimic of Smac, a pro-apoptotic protein that functions by relieving inhibitor-of-apoptosis protein (IAP)–mediated suppression of caspase activity. The compound binds to X chromosome– encoded IAP (XIAP), cellular IAP 1 (cIAP-1), and cellular IAP 2 (cIAP-2) and synergizes with both tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL) to potently induce caspase activation and apoptosis in human cancer cells. The molecule has allowed a temporal, unbiased evaluation of the roles that IAP proteins play during signaling from TRAIL and TNF receptors. The compound is also a lead structure for the development of IAP antagonists potentially useful as therapy for cancer and inflammatory diseases.
Fig. 1. C2-symmetric compound 3 is a potent Smac mimetic in vitro. (A) Chemical structures of the small molecules described in this study. (B) Fluorescence polarization assay for the interaction of Smac and mimetics with the Bir3 domain of human XIAP. A synthetic Smac peptide [AVPIAQKSEK (12)] was C-terminally labeled with Alexafluor488 (Molecular Probes), and its complex with recombinant XIAP Bir3 (residues 241 to 356) was used to evaluate competitive Bir3 domain binding by synthetic small molecules (14). (C) Polyacrylamide gel electrophoresis under nondenaturing conditions and Coomassie Blue staining were used to evaluate the binding of 3 to recombinant full-length human XIAP. XIAP (5 µM) and Smac (8 µM) were incubated for 30 min at 37°C with or without prior treatment with varying amounts of 3. Asterisks indicate that compound alone (40 µM) was present along with XIAP in lanes 3, 10, and 12. (D) Time course comparison of caspase 3 activation by recombinant Smac and small molecule mimetics. HeLa S100 was activated with 1 mM dATP. Either Smac (100 nM) or a small molecule (100 nM) was then added. The onset of caspase 3 activity was monitored as a fluorogenic substrate (Ac-DEVD-AMC, CalBiochem) was cleaved in situ (rfu, relative fluorescence units). (E) Bar graph representation of the same experiment performed in (D) except with varying concentrations of Smac and compound 3. (F) Smac and compound 3 compete with glutathione S-transferase (GST)–tagged human XIAP for active caspase 9 binding. Procaspase 9 (0.9 µM) was activated with 20 nM Apaf-1, 100 nM cytochrome C, and 1 mM dATP and then incubated with recombinant GST-XIAP for 3 hours at 30°C either in the absence (lane 2) or presence (lane 3) of Smac (1 µM), compound 3 (1 µM, lane 4), or compound 4 (1 µM, lane 5). Western blots for active caspase 9 that subsequently associates with added glutathione-coated beads are shown.
Fig. 2. Compound 3 and TRAIL act synergistically to induce apoptosis in cell culture. (A) Human glioblastoma (T98G) cells were cultured in Dulbecco's minimum essential medium (DMEM) containing fetal calf serum (10%) and treated with TRAIL (50 ng/mL) alone or compound (100 nM) alone for 15 and 19 hours, respectively. When used together, the small molecule was added 4 hours before TRAIL (total incubation time, 19 hours). Cell death (% of total population) was quantified by trypan blue staining. Values represent the average of three independent experiments (error bars indicate 1 standard derivation). (B) Activation of caspase 8 and caspase 3 by 3 in combination with TRAIL. T98G cells were treated with TRAIL (50 ng/ml) alone or 3 (100 nM) alone (for 8 and 12 hours, respectively) or were treated first with various concentrations of 3 for 4 hours and then with TRAIL for 8 hours. Cell extracts were prepared and subjected to Western blot analysis with the use of antibodies specific for caspase 8 and proteolyzed PARP. Asterisk indicates cross-reactive band. (C) Affinity purification of IAP proteins using a biotinylated form of compound 3 (fig. S2). Biotinylated 3 was immobilized onto streptavidin-conjugated beads and incubated with T98G cell extracts. The recovered beads were boiled, and released proteins were resolved by gel electrophoresis. The gel was probed with antibodies to XIAP, cIAP1 and cIAP2: Lane 1, precipitation using a negative control compound. Lane 2, precipitation using biotinylated 3. Lane 3, same as lane 2, except the cell extract was treated first with 3 (5 µM) for 4 hours.
Fig. 3. Compound 3 and TNF act synergistically to induce apoptosis in cell culture. (A) HeLa cells were cultured in DMEM containing fetal calf serum (10%) and treated with TNF (10 ng/mL) alone or compound (100 nM) alone for 15 and 19 hours, respectively. When used together, the small molecule was added 4 hours before TNF (total incubation time, 19 hours). Cell death (% of total population) was quantified by trypan blue staining. Values represent the average of three independent experiments (error bars, 1 standard derivation). (B) Hela cells were treated with TNF (10 ng/ml) alone, cycloheximide (CHX, 10 µM) alone, or a combination of TNF and CHX for 15 hours. (C) (Top) Time course of caspase 8 and caspase 3 activation in Hela cells treated with TNF and/or 3. Hela cells were treated with TNF (10 ng/ml) alone, 3 (100 nM) alone, or treated with 3 for 4 hours and then with TNF. Cell extracts were made at indicated times and subjected to Western blot analysis with antibodies to caspase 8 or proteolyzed PARP. (Bottom) Time course of caspase 3 activation in Hela cells treated with TNF, CHX, or both.
