Cells originating from the same population were trasfected in order to over-express either the active HCE-WT-HA, the GTase-defective HCE-K294A-HA mutant, the GFP control protein or no protein. They were submitted to concentrations of 0 mM, 40 mM or 120 mM of mizoribine. All cell lines treated with mizoribine showed a global reduction in reporter protein expression when compared with untreated cells. This expected effect is likely due to partial guanosine pool depletion induced by IMPDH inhibition. Interestingly, the reduction in transcription and translation of the reporter was significantly less severe only in cells over-expression HCE-WT-HA for both mizoribine concentrations. The ability of HCE-WT-HA over-expression to partially rescue the luciferase expression in the presence of mizoribine ABT-199 demonstrates that HCE is one of the mizoribine pharmacological targets. Furthermore, the inability of the GTase defective mutant HCE-K294A-HA to rescue the reporter expression under mizoribine treatment further demonstrates, in agreement with our in vitro results, that in a cellular context it is the GTase activity of HCE that is targeted by MZP. Although indirect, this is strong evidence that mizoribine is able to impair capping in a cellular environment. As expected, capping could not be fully inhibited in cellulo at mizoribine concentrations of 40�C120 mM, which is approximately the in vitro IC50 of 80 mM. Despite its oral bioavailability and its low binding to serum proteins, mizoribine was not expected to reach intracellular concentration higher then its IC50. Of notice, the effect of mizoribine on cellular capping was however greater than anticipated. We hypothesize that the competition between HCE and Xrn2 for the nascent mRNA 59 end could explain the potency of MZP in cellulo, as slowing the capping activity could be sufficient to shift the balance towards quality control take-over and reduce downstream protein expression. What is the exact contribution of the capping apparatus inhibition to the global mizoribine mechanism of action? This question has yet to be addressed, but the immunosuppressive effect of mizoribine on T-cell is mainly mediated by GTP depletion and might be exacerbated by the reduction of mRNA capping and downstream cap-dependent translation. Our study clearly demonstrates that the therapeutic agent mizoribine monophosphate SB431542 301836-41-9 inhibits the human RNA guanylyltransferase in vitro and impairs mRNA capping in cellulo. Estrogen receptor negative breast cancer types are generally more aggressive and prone to metastasize. The absence of Estrogen receptor-alpha correlates with hormone-independent growth of these mammary tumor cells and causes uncontrolled proliferation and insensitivity to anti-hormonal treatments. In ERa-negative cell lines, a subset of genes is epigenetically silenced, while the majority of genes involved in cell cycle control and proliferation are constitutively expressed. Aberrant gene expression is frequently the result of chromatin modifications and composition, including histone posttranslational modifications and/or incorporation of histone variants. In particular, deregulation of enzyme complexes responsible for histone acetylation and deacetylation can be associated with breast cancer progression and an increase in tumor malignancy. Thus, compounds that change chromatin modifications are a promising anti-cancer approach.

In addition to inhibiting cell cycle progression, BEZ235 caused apoptosis in two of six cell lines. The inhibition of cell cycle Compound Library progression is a known effect of BEZ235, even at lower doses. However, apoptosis appears in only some cancer cell lines and is more apparent at higher doses of BEZ235. BEZ235 efficiently inhibited mTORC1, a molecule AB1010 controlling both cell cycle and apoptosis that might lead to cell cycle arrest and apoptosis in KAT4C and KAT18. Understanding the mechanisms through which BEZ235 contributes to apoptotic cell death will require further study. ATC is by far the most aggressive of the four major histologic types of thyroid cancer. Chemotherapy has been applied to treat patients with ATC with response rates around 20 to 50%. Novel strategies to improve outcomes are needed. Among three combination therapy regimens, BEZ235 combined with paclitaxel had the best synergistic effect in four ATC cell lines. Cancer cells with activation of PI3K/mTOR signaling are more resistant to paclitaxel, and the co-administration of PI3K/mTOR inhibitors with paclitaxel improves therapeutic effects. This finding is of clinical relevance since paclitaxel, a microtubule stabilizer, has shown to achieve a 53% response rate in patients with ATC in a phase II clinical trial. The combination of BEZ235 with a microtubule depolymerizing drug vincristine also revealed promising effect in the treatment of sarcoma. Our data showed that the combinational effects of BEZ235 with inhibitors of DNA topoisomerase type I or type II in treating ATC were largely antagonistic. Similar antagonistic effects of inhibition both topoisomerase activity and PI3K/AKT pathway have been observed in ovarian cancer cells. Cells in S phase are more susceptible to topoisomearse inhibitors. BEZ235-induced accumulation of cells at G0/G1 phase may therefore reduce the therapeutic advantage of concurrent therapy with topoisomerase inhibitors, explaining the unfavorable combination effects of BEZ235 with irinotecan and etoposide. Daily treatment of BEZ235 significant retarded 8505C xenograft tumor growth during the therapeutic period. The inhibitory effect was less prominent after the discontinuation of therapy, suggesting that prolonged treatment may be necessary to maintain therapeutic efficacy. BEZ235 significantly degraded caspase-3 in 8505C xenograft tumors, indicating this compound may induce apoptosis in vivo. After discontinuation of BEZ235, the volume of 8505C xenograft tumors subsequently increased. This enlargement of tumor following cessation of therapy might be due to normalization of cell size and resumption of cell proliferation, as mTOR and its downstream proteins S6 Kinase 1 and 4E-BP1 play pivotal roles in this respect. No significant weight loss or illness was observed during study period, suggesting that this therapy may have a promising safety profile. A prior report demonstrated that the presence of genetic alterations of PTEN, PIK3CA and AKT1 correlated well with sensitivity to an AKT inhibitor, but had weaker correlations with an mTOR inhibitor. This discrepency suggests that mTOR activity does not depend solely on PI3K/AKT activity. The reported data of genetic alterations in 8 thyroid cancer cell lines was summarized. Limited genetic changes on RAS/RAF/ERK and PI3K/mTOR pathways were identified.

RTase and GTase domains since the abolition of the RTase domain EX 527 HDAC inhibitor reduces HCE MZP susceptibility. In order to gain additional details on the MZP main binding site, molecular docking could be used. Unfortunately, the structure of both the RTase and the GTase domain are separately available, but the structure of the full-length protein is still not available. Nevertheless, we ran a molecular docking experiment of MZP on the GTase domain from HCE. The lack of RTase domain, which is predicted to participate in MZP binding, and the moderate flexibility of the N-terminal domain, which introduces a structural incertitude, does not allow for definitive conclusions to be reached but it is interesting to note that, in preliminary experiments, MZP favorably docks on the N-terminal region of the HCE GTase. Together, our results reveal that MZP inhibits the HCE GTase activity with a 5-to 25-fold specificity in comparison to other GTases. Although more work is yet required to confirm our hypothesis, these results raise the possibility that the GTase inhibition could be mediated by a conformational change hindrance upon binding of MZP to an allosteric binding site that is speculated to reside near the RTase-GTase inter-domain. Nevertheless, mizoribine is one of the first compounds to demonstrate a certain degree of specificity toward a single GTase, despite the high degree of conservation of this crucial family of enzyme. MZP displays a higher in vitro inhibition potency for the GTase reaction in comparison to the complete RNA capping reaction. This may simply be due to our experimental conditions where the RTase activity of HCE, which is partially inhibited by free Mg2+, becomes the rate-limiting step. However, in cellulo the RTase harbors a higher BEZ235 turnover rate than the GTase, which catalyzes the limiting step in RNA capping. In a cellular context we expect the efficiency of MZP to be dictated solely by its interaction with the GTase. Historically, very few GTase inhibitors have been developed, neither as scientific tools nor as therapeutic agents. More recently however, novel GTase inhibitors have been discovered. They include the allosteric inhibitor mycophenolic acid, the pyrophosphate analog foscarnet which acts as a product inhibitor, and ribavirin triphosphate, a GTP analog that is transferred to acceptor RNAs by GTase, leading to stable but inefficiently translated pseudo-capped RNA. The current study identifies MZP as a novel allosteric GTase inhibitor, which is speculated to block a crucial conformational change. The GTase activity being the ratelimiting step of the essential capping apparatus, all these GTase inhibitors are promising lead candidates for the development of novel selective capping inhibitors and lead the way to a new class of anti-cancer, antifungal, and antiviral drugs. What is the biological relevance of the present finding? Numerous studies have demonstrated the potency of MZP to inhibit the cellular IMPDH and to lower the intracellular guanosine nucleotide pool thereby limiting cell growth, but none have addressed its impact on the capping apparatus. Monitoring the capping efficiency in living cells is a great challenge as the cellular quality control machinery degrades unsuccessfully capped mRNAs. Since proper capping is crucial for mRNA transcription, export, stability and translation, it is possible to monitor the capping efficiency based on the translation of a reporter protein.