Mtor small molecule activator

To further investigate the underlying mechanism of FLJ action, we used bioinformatics to predict the functioning modes of FLJ When adding mutant substrates for MIR , the decreased luciferase activity was abrogated Fig.

Figure 8. Among the putative targets of MIR , we concentrated on ATG13 because it could bind with MIR with the highest score and is a key gene in the regulation of autophagy. Figure 9. Rapamycin inhibited the level of p-ATG13 as compared with normal controls Fig. The signaling pathways mediated by MTOR regulate cell proliferation, growth, and metabolism. So far, no chemical activators of MTOR have been found.

The molecule of 3BDO is smaller than that of rapamycin, so we deduced that 3BDO may occupy rapamycin binding sites easier than rapamycin. Although researchers have made many advances in defining the roles of the MTOR signal pathway in autophagy, the current challenge is to find downstream components involved in this signaling. Changes in the transcript level of FLJ have been observed in many microarray assays, 17 - 26 but its function and mechanism were unknown.

TIA1, which can bind DNA and RNA, is involved in regulating gene expression at both transcriptional and posttranscriptional levels in eukaryotic cells. Furthermore, we first investigated the possible pattern of FLJ processing in this study in light of several considerations. Many studies show that TIA1 could affect cell proliferation, apoptosis, angiogenesis, invasion, and metastasis, and evasion of immune recognition. The data suggest that large numbers of uaRNA in human, mouse, and fly undergo post-transcriptional processing.

Therefore, we draw a tentative conclusion that FLJ is processed by post-transcriptional cleavage. Thus, in our next study, we will conduct the luciferase activity assay to detect whether any promoters exist in the internal or vicinity of FLJ and the capped analysis of gene expression CAGE assay 44 to further demonstrate the post-transcriptional processing of FLJ At present, finding new noncoding transcripts associated with miRNA and its targeted gene functions is advantageous, the importance of which has been revealed recently.

Among the various predicted targets of MIR , we concentrated on ATG13 , because it encodes for the protein with an essential role in autophagy. Our results are consistent with the previous conclusion that decoy lncRNAs modulate gene expression by binding with miRNAs. ATG13, which associates with ULK1 and RB1CC1 to form a stable protein complex, localizes on the phagophore membrane and is essential for autophagosome formation and then autophagy initiation. This process is involved in autophagy regulation.

Our findings highlight the role and action mechanism of this lncRNA in autophagy, and 3BDO might be a potential drug to treat diseases caused by disorders of autophagy.

Figure Cells up to passage 10 were used for experiments. All docking conformations were ranked according to the CScore, and the best-ranked results CScore 5 was checked visually. Ser phosphorylation was detected by western blot assay with Ser phosphorylation antibody Santa Cruz Biotechnology, sc The microarray process was as described. Total RNA was extracted as described previously.

Then vectors were transformed into Trans1-T1-competent cells. Blue white spot screening was used to detect recombinant clones. Clones were cultured in LB liquid medium overnight and plasmids were extracted. Immunofluorescence assay was performed as described. Cells were rinsed 3 times with 0. Transient transfection involved the LipofectamineTM method Invitrogen, Cells were cultured for another 24 h for the desired assays.

The immunoreactive bands were developed with enhanced chemiluminessence Thermo Fisher, The relative quantity of proteins was analyzed by use of ImageJ. Cells were pelleted by centrifugation for 10 min at 3, rpm and resuspended with lysis buffer, then chromatin was sheared with the appropriate conditions. The position 1 is the TIA1 binding sequence, and position 2 is the negative control sequence. A mutant construct was generated by a similar approach.

Luciferase activity assays involving a dual-luciferase reporter system were conducted according to the protocol provided by Promega Promega, E Cells were cotransfected with Dual-Luciferase containing firefly and Renilla luciferase reporter constructs and corresponding miRNA mimics, 10 and 40 nM, by use of Lipofectamine After a shaking for 30 min, Renilla luciferase activities were measured in the same tube. Luciferase activities after different treatments were to that of Renilla luciferase activities.

RNA was isolated with the Trizol reagent method. RNA was quantified by use of the NanoDrop. U6 was the control for small RNAs. The final concentrations of the mimics and NC were 10 or 50 nM. Mean values were derived from at least 3 independent experiments. National Center for Biotechnology Information , U. Journal List Autophagy v. Published online Apr 1. Author information Article notes Copyright and License information Disclaimer.

This article has been cited by other articles in PMC. Associated Data Supplementary Materials Additional material. Abstract MTOR, a central regulator of autophagy, is involved in cancer and cardiovascular and neurological diseases. Open in a separate window. Immunofluorescence assay Immunofluorescence assay was performed as described. Luciferase activity assays Luciferase activity assays involving a dual-luciferase reporter system were conducted according to the protocol provided by Promega Promega, E Supplementary Material Additional material Click here to view.

Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Reference 1. The emerging importance of autophagy in pulmonary diseases. Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev. Targeting mammalian target of rapamycin mTOR for health and diseases. Drug Discov Today. Cancer Lett. Weichhart T. Mammalian target of rapamycin: a signaling kinase for every aspect of cellular life. Methods Mol Biol. Alayev A, Holz MK.

J Cell Physiol. Rapamycin and 3-methyladenine regulate apoptosis and autophagy in bone-derived endothelial progenitor cells. Chin Med J Engl ; — Hypoinsulinaemia, glucose intolerance and diminished beta-cell size in S6K1-deficient mice. Upregulation of the mammalian target of rapamycin complex 1 pathway by Ras homolog enriched in brain in pancreatic beta-cells leads to increased beta-cell mass and prevention of hyperglycemia. Laplante M, Sabatini DM. Chem Res Toxicol.

A butyrolactone derivative suppressed lipopolysaccharide-induced autophagic injury through inhibiting the autoregulatory loop of p8 and p53 in vascular endothelial cells. Int J Biochem Cell Biol. J Alzheimers Dis. Spring DR. Chemical genetics to chemical genomics: small molecules offer big insights. Chem Soc Rev.

Molecular signature of smoking in human lung tissues. Cancer Res. For example, temsirolimus and everolimus exhibit tumor-suppressive effects in vivo. Both temsirolimus and everolimus have been used to treat advanced renal cell carcinoma RCC in the clinic. Moreover, everolimus is prescribed for treating pancreatic neuroendocrine tumors and advanced breast cancer. Given the specificity of the drug and its potency in anticancer activity in many preclinical models, it was hard to imagine the need for new types of mTOR inhibitors.

However, the clinical application of rapalogs in cancer treatment has so far met with limited success. Several selective-mTOR inhibitors have been produced such as XL, Ku and torkinib , and some of these agents are in the early stages of clinical trials.

Tumors that are addicted to the mTOR signaling pathway may be sensitive to this kind of inhibitor. In , a series of selective mTOR inhibitors were reported. Additional mTOR inhibitors were also developed subsequently that are in general more potent and selective for mTOR inhibition.

Therefore, it can be used to treat glioma and reverse temozolomide resistance. Multiple studies in distinct cancer lineages have revealed that mTORC1 inhibition, while not sufficient in most cases, is likely necessary to achieve a clinical response to drugs that target the primary oncogenic pathway in a given cancer.

Using genetically-defined cancer cell lines, tumor models, and patient samples, these studies have all arrived at the same conclusion: sustained mTORC1 signaling following treatment with a targeted therapeutic is strongly associated with both innate and acquired resistance to that therapeutic.

Thus, while mTORC1 inhibitors appear to be limited in their use as single-agent therapies, there is growing evidence that they may be effective in combinatorial approaches to cancer treatment, specifically when used with targeted therapeutics. The discovery of TOR in yeast and mTOR in mammals is a fundamental breakthrough in understanding cell and organism growth, metabolism, and diseases. In recent years, several magnificent progresses have been achieved in our understanding of the signaling pathways with the central role of mTOR molecules.

Deregulation of different elements of the mTOR pathway has been reported in many types of cancers. The identification of the critical roles of mTOR and its regulators in tumorigenesis has driven the development of the ever-growing list of mTOR inhibitors.

While some of the mTOR inhibitors have been approved to treat cancer patients, more mTOR inhibitors are under check to fulfill their promise for cancer therapy. Small Molecule Compounds and mTOR Signaling Pathway Intracellular signaling pathways collect and transduce extracellular signals into cells, which affect cellular proliferation, growth, and metabolism.

Compounds related to the mTOR signaling pathway:. Torin 1.