Administration of Gapmer-type Antisense Oligonucleotides Targeting γ-Glutamylcyclotransferase Suppresses the Growth of A549 Lung Cancer Xenografts.

BACKGROUND/AIM: γ-Glutamyl cyclotransferase (GGCT) is up-regulated in various cancer types, including lung cancer. In this study, we evaluated efficacy of gapmer-type antisense oligonucleotides (ASOs) targeting GGCT in an A549 lung cancer xenograft mouse model and studied their mechanisms of action. MATERIALS AND METHODS: GGCT was inhibited using GGCT-ASOs and cell proliferation was evaluated by dye exclusion test. Western blot analysis was conducted to measure expression of GGCT, p21, p16 and p27, phosphorylation of AMP-activated protein kinase, and caspase activation in A549 cells. Induction of apoptosis and up-regulation of reactive oxygen species were assessed by flow cytometry using annexin V staining and 2',7'-dichlorodihydrofluorescein diacetate dye, respectively. RESULTS: GGCT-ASOs suppressed GGCT expression in A549 cells, inhibited proliferation, and induced apoptosis with activation of caspases. GGCT-ASOs also increased expression of cell-cycle regulating proteins, phospho-AMPK and ROS levels. Systemic administration of GGCT-ASOs to animals bearing A549 lung cancer xenografts showed significant antitumor effects without evident toxicity. CONCLUSION: GGCT-ASOs appear to be promising as novel cancer therapeutic agents.

Discovery of C13-Aminobenzoyl Cycloheximide Derivatives that Potently Inhibit Translation Elongation.

Employed for over half a century to study protein synthesis, cycloheximide (CHX, 1) is a small molecule natural product that reversibly inhibits translation elongation. More recently, CHX has been applied to ribosome profiling, a method for mapping ribosome positions on mRNA genome-wide. Despite CHX's extensive use, CHX treatment often results in incomplete translation inhibition due to its rapid reversibility, prompting the need for improved reagents. Here, we report the concise synthesis of C13-amide-functionalized CHX derivatives with increased potencies toward protein synthesis inhibition. Cryogenic electron microscopy (cryo-EM) revealed that C13-aminobenzoyl CHX (8) occupies the same site as CHX, competing with the 3' end of E-site tRNA. We demonstrate that 8 is superior to CHX for ribosome profiling experiments, enabling more effective capture of ribosome conformations through sustained stabilization of polysomes. Our studies identify powerful chemical reagents to study protein synthesis and reveal the molecular basis of their enhanced potency.

Involvement of FKBP6 in hepatitis C virus replication.

The chaperone system is known to be exploited by viruses for their replication. In the present study, we identified the cochaperone FKBP6 as a host factor required for hepatitis C virus (HCV) replication. FKBP6 is a peptidyl prolyl cis-trans isomerase with three domains of the tetratricopeptide repeat (TPR), but lacks FK-506 binding ability. FKBP6 interacted with HCV nonstructural protein 5A (NS5A) and also formed a complex with FKBP6 itself or FKBP8, which is known to be critical for HCV replication. The Val(121) of NS5A and TPR domains of FKBP6 were responsible for the interaction between NS5A and FKBP6. FKBP6 was colocalized with NS5A, FKBP8, and double-stranded RNA in HCV-infected cells. HCV replication was completely suppressed in FKBP6-knockout hepatoma cell lines, while the expression of FKBP6 restored HCV replication in FKBP6-knockout cells. A treatment with the FKBP8 inhibitor N-(N', N'-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication. HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue. These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A.

FKBPs in bacterial infections.

BACKGROUND: FK506-binding proteins (FKBPs) contain a domain with peptidyl-prolyl-cis/trans-isomerase (PPIase) activity and bind the immunosuppressive drugs FK506 and rapamycin. FKBPs belong to the immunophilin family and are found in eukaryotes and bacteria. SCOPE OF REVIEW: In this review we describe two major groups of bacterial virulence-associated FKBPs, the trigger factor and Mip-like PPIases. Moreover, we discuss the contribution of host FKBPs in bacterial infection processes. MAJOR CONCLUSIONS: Since PPIases are regarded as alternative antiinfective drug targets we highlight current research strategies utilizing pipecolinic acid and cycloheximide derivatives as well as substrate based inhibitors. GENERAL SIGNIFICANCE: The current research strategies suggest a beneficial synergism of drug development and basic research. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Structure-activity relationship of 9-methylstreptimidone, a compound that induces apoptosis selectively in adult T-cell leukemia cells.

We previously reported that 9-methylstreptimidone, a piperidine compound isolated from a culture filtrate of Streptomyces, induces apoptosis selectively in adult T-cell leukemia cells. It was screened for a compound that inhibits LPS-induced NF-kappaB and NO production in mouse macrophages. However, 9-methystreptimidone is poorly obtained from the producing microorganism and difficult to synthesize. Therefore, in the present research, we studied the structure-activity relationship to look for new selective inhibitors. We found that the structure of the unsaturated hydrophobic portion of 9-methylstreptimidone was essential for the inhibition of LPS-induced NO production. Among the 9-methylstreptimidone-related compounds tested, (+/-)-4,alpha-diepi-streptovitacin A inhibited NO production in macrophage-like cells as potently as 9-methylstreptimidone and without cellular toxicity. Moreover, this compound selectively induced apoptosis in adult T-cell leukemia MT-1 cells.

Novel fluorescent cycloheximide derivatives for the imaging of protein synthesis.

Cycloheximide (CHX) is one of the most interesting protein synthesis inhibitors. For this reason, fluorescent derivatives of CHX could find useful applications in cell biology. We report the successful synthesis of a set of novel fluorescent derivatives of CHX. The effect of different functional groups on the biological activity of CHX was studied upon their modification through suitable strategies, i.e., acetylation of the hydroxyl group and reductive amination of the ketone group. The first route induced a complete loss of biological activity, while the second approach allowed a retained inhibition of protein synthesis, as demonstrated by in vitro translation assays. Various fluorescent dyes for reductive amination were tested (i.e., ANTS, APTS, and Rhodamine-123), and the success of the syntheses was demonstrated by diverse analytical techniques. Cycloheximide labeling with fluorescent dyes is a promising approach for developing fluorescence reporters for various applications, both in vitro (fluorescence spectroscopy) and in vivo (live imaging).

[Antiviral activities of cycloheximide and its derivatives].

Cycloheximide (CHX) inhibits protein synthesis in most eukaryotic cells and it is a well-known tool commonly used in biochemical research. In this paper, the antiviral spectrum of CHX against several DNA and RNA viruses have been evaluated. CHX showed strong inhibitory activities against several RNA viruses such as HIV-1, influenza viruses, coxsackie B virus, enterovirus (EV71) and several DNA viruses such as HSV and HCMV. Especially the strong inhibitory activities of CHX against coxsackie B virus and enterovirus caught our attention, since effective drugs available in clinic are limited. The SAR of CHX derivatives also has been discussed in the paper. The hydroxyl group at C-2' and carbonyl group at C-2" of CHX are essential for its antiviral activity. And modification to these groups results its derivatives' antiviral activities reduced or lost.

Caspase-8 mediates mitochondrial release of pro-apoptotic proteins in a manner independent of its proteolytic activity in apoptosis induced by the protein synthesis inhibitor acetoxycycloheximide in human leukemia Jurkat cells.

The cysteine protease caspase-8 plays an essential role in apoptosis induced by death receptors. The protein synthesis inhibitor acetoxycycloheximide (Ac-CHX) has been previously shown to induce rapid apoptosis mediated by the release of cytochrome c in human leukemia Jurkat cells. In this study, the novel molecular mechanism that links caspase-8 to the mitochondrial release of pro-apoptotic proteins has been identified. Jurkat cells deficient in caspase-8 were more resistant to Ac-CHX than wild-type Jurkat cells and manifested decreased apoptosis induction and caspase activation as well as inefficient release of cytochrome c, Smac/DIABLO, and AIF into the cytosol. In contrast to Fas ligand stimulation, the general caspase inhibitor barely prevented the mitochondrial release of these pro-apoptotic proteins in Ac-CHX-treated cells, suggesting that caspase-8 activity is dispensable for triggering the mitochondrial pathway in Ac-CHX-induced apoptosis. Consistent with this notion, caspase-8-deficient Jurkat cells reconstituted with catalytically inactive caspase-8 became sensitive to Ac-CHX and exhibited apoptosis, caspase activation, the liberation of pro-apoptotic proteins into the cytosol, and Bak conformational change as efficiently as wild-type Jurkat cells. Unlike caspase-3, -6, -7, and -9, a small but significant portion of caspase-8 was found to localize in mitochondria before and after exposure to Ac-CHX. These results clearly demonstrate that caspase-8 is able to mediate the mitochondrial release of pro-apoptotic proteins in a manner independent of its proteolytic activity in Ac-CHX-induced apoptosis.

ERK and p38 MAP kinase are involved in downregulation of cell surface TNF receptor 1 induced by acetoxycycloheximide.

Tumor necrosis factor (TNF)-alpha activates the nuclear factor kappaB (NF-kappaB) signaling pathway. The protein synthesis inhibitor cycloheximide (CHX) and its structural derivative acetoxycycloheximide (Ac-CHX) have been recently shown to block the TNF-alpha-induced activation of NF-kappaB via ectodomain shedding of TNF receptor 1 (TNF-R1) in human lung carcinoma A549 cells. In this study, we show that ERK and p38 MAP kinase are involved in the downregulation of cell surface TNF-R1 upon exposure to Ac-CHX and the subsequent inhibition of TNF-alpha-induced NF-kappaB activation. Ac-CHX was capable of promoting the sustained activation of ERK, JNK, and p38 MAP kinase. Treatment with the MEK inhibitor U0126 and the p38 MAP kinase inhibitor SB203580, but not the JNK inhibitor SP600125, reversed the diminished expression of cell surface TNF-R1 as well as the blockade of TNF-alpha-induced IkappaBalpha degradation in Ac-CHX-treated cells. Our results indicate that Ac-CHX triggers the downregulation of cell surface TNF-R1 via the activation of ERK and p38 MAP kinase, thereby preventing activation of the NF-kappaB signaling pathway by TNF-alpha.

Ectodomain shedding of TNF receptor 1 induced by protein synthesis inhibitors regulates TNF-alpha-mediated activation of NF-kappaB and caspase-8.

The transcription factor nuclear factor kappaB (NF-kappaB) plays a major role in the inducible resistance to death receptor-mediated apoptosis. It has been established that the protein synthesis inhibitor cycloheximide (CHX) sensitizes many types of cells to tumor necrosis factor (TNF)-alpha-induced apoptosis, mainly due to its ability to block de novo synthesis of cellular FLICE-inhibitory protein (c-FLIP). Nevertheless, we have surprisingly found that CHX, as well as its structural analogue acetoxycycloheximide (Ac-CHX), prevents TNF-alpha-mediated activation of NF-kappaB and caspase-8 in human lung carcinoma A549 cells. Both CHX and Ac-CHX reduced the expression of cell surface TNF receptor 1 (TNF-R1) in a dose-dependent manner, while Ac-CHX was approximately 100-fold more effective than CHX. Consistent with this observation, Ac-CHX induced the proteolytic cleavage of TNF-R1 and its release into the culture medium. CHX and Ac-CHX profoundly decreased constitutive and inducible expression of c-FLIP, whereas these compounds potentiated TNF-alpha-induced caspase-8 activation only when metalloprotease inhibitors were present. Thus, our results indicate that ectodomain shedding of TNF-R1 induced by protein synthesis inhibitors regulates TNF-alpha-mediated activation of NF-kappaB and caspase-8.

The specific FKBP38 inhibitor N-(N',N'-dimethylcarboxamidomethyl)cycloheximide has potent neuroprotective and neurotrophic properties in brain ischemia.

FK506 and FK506-derived inhibitors of the FK506-binding protein (FKBP)-type peptidylprolyl cis/trans-isomerases (PPIase) display potent neuroprotective and neuroregenerative properties in various neurodegeneration models, showing the importance of neuroimmunophilins as targets for the treatment of acute and chronic neurodegenerative diseases. However, the PPIase activity targeted by active site-directed ligands remains unknown so far. Here we show that neurotrophic FKBP ligands, such as GPI1046 and N-[methyl(ethoxycarbonyl)]cycloheximide, inhibit the calmodulin/Ca(2+) (CaM/Ca(2+))-regulated FKBP38 with up to 80-fold higher affinity than FKBP12. In contrast, the non-neurotrophic rapamycin inhibits FKBP38.CaM/Ca(2+) 500-fold less affine than other neuroimmunophillins. In the context of the high expression of FKBP38 in neuroblastoma cells, these data suggest that FKBP38.CaM/Ca(2+) inhibition can mediate neurotrophic properties of FKBP ligands. The FKBP38-specific cycloheximide derivative, N-(N',N'-dimethylcarboxamidomethyl)cycloheximide (DM-CHX) was synthesized and used in a rat model of transient focal cerebral ischemia. Accordingly, DM-CHX caused neuronal protection as well as neural stem cell proliferation and neuronal differentiation at a dosage of 27.2 mug/kg. These effects were still dominant, if DM-CHX was applied 2-6 h post-insult. In parallel, sustained motor behavior deficits of diseased animals were improved by drug administration, revealing a potential therapeutic relevance. Thus, our results demonstrate that FKBP38 inhibition by DM-CHX regulates neuronal cell death and proliferation, providing a promising strategy for the treatment of acute and/or chronic neurodegenerative diseases.

Acetoxycycloheximide (E-73) rapidly induces apoptosis mediated by the release of cytochrome c via activation of c-Jun N-terminal kinase.

Cycloheximide (CHX) is an inhibitor of protein synthesis and commonly used to modulate death receptor-mediated apoptosis or to induce apoptosis in a number of normal and transformed cells. In this study we show that a close structural derivative of CHX, acetoxycycloheximide (E-73) induced rapid processing of procaspases and subsequent apoptosis with much higher efficacy than CHX in human leukemia Jurkat T cells. E-73 induced the release of cytochrome c from mitochondria even in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone. The Bcl-2 family protein Bcl-x(L) suppressed cytochrome c release as well as processing of procaspases-3, -8, and -9 in E-73-treated cells. In Jurkat T cells transfected with the caspase-8 modulator FLIP(L), E-73 still induced activation of procaspase-3 and subsequent apoptosis, suggesting that the caspase-8 activity is dispensable for apoptosis. In contrast to CHX, E-73 drastically induced activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 MAP kinase. Inhibitory profiles of small-molecular kinase inhibitors revealed that JNK activation was critical for induction of cytochrome c release in E-73-induced apoptosis. Thus, our present results demonstrate that E-73, unlike CHX, induces strong activation of the JNK pathway and triggers rapid apoptosis mediated by the release of cytochrome c.

E-73, an acetoxyl analogue of cycloheximide, blocks the tumor necrosis factor-induced NF-kappaB signaling pathway.

Proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-1 activate the NF-kappaB signaling pathway which induces the expression of a variety of genes such as the encoding intercellular adhesion molecule (ICAM)-1. We have found that E-73, an acetoxyl analogue of cycloheximide, specifically blocks TNF-induced ICAM-1 expression even at concentrations unable to affect protein synthesis. By contrast, cycloheximide inhibited both TNF- and IL-1-induced ICAM-1 expression primarily due to the blockage of protein synthesis. The nuclear translocation of NF-kappaB as well as the IkappaB degradation induced by TNF, but not by IL-1, was significantly prevented by E-73. These observations suggest that E-73 blocks the TNF-induced NF-kappaB signaling pathway upstream of IkappaB degradation.

Synthesis and cytotoxic evaluation of cycloheximide derivatives as potential inhibitors of FKBP12 with neuroregenerative properties.

On the basis of the new finding that the protein synthesis inhibitor cycloheximide (1, 4-[2-(3, 5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]-2,6-piperidinedione) is able to competitively inhibit hFKBP12 (K(i) = 3.4 microM) and homologous enzymes, a series of derivatives has been synthesized. The effect of the compounds on the activity of hFKBP12 and their cytotoxicity against eukaryotic cell lines (mouse L-929 fibroblasts, K-562 leukemic cells) were determined. As a result, several less toxic or nontoxic cycloheximide derivatives were identified by N-substitution of the glutarimide moiety and exhibit IC(50) values in the range of 22.0-4.4 microM for inhibition of hFKBP12. Among these compounds cycloheximide-N-(ethyl ethanoate) (10, K(i) = 4.1 microM), which exerted FKBP12 inhibition to an extent comparable to that of cycloheximide (1), was found to cause an approximately 1000-fold weaker inhibitory effect on eukaryotic protein synthesis (IC(50) = 115 microM). Cycloheximide-N-(ethyl ethanoate) (10) was able to significantly speed nerve regeneration in a rat sciatic nerve neurotomy model at dosages of 30 mg/kg.

Syntheses and biological activities of (+/-)-streptovitacin A and E-73.

(+/-)-Streptovitacin A (1) and its stereoisomers were synthesized by an aldol reaction of (+/-)-2,4-dimethyl-4-trimethylsiloxy-1-cyclohexanones (4b and 9) with 4-(2-oxoethyl)-2,6-piperidinedione (5). E-72 (2) was derived from synthetic 1. (+/-)-1 showed moderate growth inhibition against fungi and lettuce seeds.

The activity of bleomycin and modifiers of its clastogenic effect on the interphase chromatin of Indian muntjak fibroblasts.

Premature chromosome condensation was induced in Indian muntjak fibroblasts after exposure of the cells to bleomycin. Further experiments were devoted to the interaction of anticlastogens and a repair inhibitor, streptovitacin A. Chromosomal aberrations due to bleomycin treatment were S-phase-independently visible in the G1 and G2 phase of the cell cycle. For premature chromosome condensation experiments, a 100-fold lower concentration of the mutagen produced a similar extent of chromosome damage as in metaphase studies. Additional exposure to the anticlastogens beta-aminoethylisothiouronium or N-acetylcysteine revealed differences between corresponding interphase and metaphase effects and between different exposure conditions. Streptovitacin A, known as an inhibitor of protein synthesis, acted like an anticlastogen in the G2 phase of the cell cycle. Our studies show that the premature chromosome condensation technique offers various qualitative insights into primary processes of mutagenicity and antimutagenicity, but requires further improvement and careful choice of the cell system for study.

Cycloheximide analogues as potential anticonvulsants.

A series of 22 cycloheximide analogues in which the substituents on the cycloheximide ring and imide nitrogen were varied, the glutarimide ring was changed to a succinimide ring, and the ring and/or side-chain oxygens were present as ketone and/or alcohol groups were prepared and sent to the Anticonvulsant Drug Development program of the National Institute of Neurological and Communicative Disorders and Strokes for evaluation as anticonvulsants. Three compounds, namely cycloheximide (1a), 2-methyl dione 2c, and dihydrocycloheximide (4a), were further evaluated in Phase II testing for quantification of maximum activity with the latter eventually progressing to Phase IV and Phase VI screens.