Nuovi coniugati fra acido azelaico ed azaeterocicli: sintesi e valutazione dell'attività biologica

The well-known antiproliferative properties of the 9-hydroxystearic acid (9-HSA) on human colon cancer cells (HT-29 cell line) have inspired this thesis work in order to obtain new derivatives maintaining the C1-C8 chain of the HSA linked to an heterocyclic moiety at the C-9 carbon atom and to investigate their biological activity. First, thiazoles, thiadiazoles and benzothiazoles, that are compounds of interest in many fields for their biological activities, have been introduced through an amide bond starting from their 2-amino precursors. The products have been obtained by treatment with methyl 9-chloro-9-oxononanoate according to a Schotten-Baumann type reaction. The acylation reaction occurred at the endocyclic nitrogen atom of the heterocycle, as ascertained through NOESY-1D experiment. After, methyl 9-chloro-9-oxononanoate was reacted with indole, N-methylindole, and triptamine giving a serie of new indole derivatives. Finally, the biological activity of some compounds has been tested through assays on HT-29 cancer cells and bacterial and fungal microorganisms; docking calculations have also been performed to evaluate the possible interactions with the active site of histone deacetylase, which are molecular targets of the 9-HSA.

Structural modification of DNA--a therapeutic option in SLE?

Systemic lupus erythematosus (SLE) is an autoimmune disease that affects multiple organs, with glomerulonephritis representing a frequent and serious manifestation. SLE is characterized by the presence of various autoantibodies, including anti-DNA antibodies that occur in approximately 70% of patients with SLE and which contribute to disease pathogenesis. Consequently, immunosuppressive therapies are applied in the treatment of SLE to reduce autoantibody levels. However, increasing evidence suggests that DNA--especially double--stranded DNA-constitutes an important pathogenic factor that is able to activate inflammatory responses by itself in autoimmune diseases. Therefore, modifying the structure of DNA to reduce its pathogenicity might be a more targeted approach for the treatment of SLE than immunosuppression. This article presents information in support of this strategy, and discusses the potential methods of DNA structure manipulation--in light of data obtained from mouse models of SLE--including topoisomerase I inhibition, administration of DNase I, or modification of histones using heparin or histone deacetylase inhibitors.

Mice generated by in vitro fertilization exhibit vascular dysfunction and shortened life span

Children conceived by assisted reproductive technologies (ART) display a level of vascular dysfunction similar to that seen in children of mothers with preeclamspia. The long-term consequences of ART-associated vascular disorders are unknown and difficult to investigate in healthy children. Here, we found that vasculature from mice generated by ART display endothelial dysfunction and increased stiffness, which translated into arterial hypertension in vivo. Progeny of male ART mice also exhibited vascular dysfunction, suggesting underlying epigenetic modifications. ART mice had altered methylation at the promoter of the gene encoding eNOS in the aorta, which correlated with decreased vascular eNOS expression and NO synthesis. Administration of a deacetylase inhibitor to ART mice normalized vascular gene methylation and function and resulted in progeny without vascular dysfunction. The induction of ART-associated vascular and epigenetic alterations appeared to be related to the embryo environment; these alterations were possibly facilitated by the hormonally stimulated ovulation accompanying ART. Finally, ART mice challenged with a high-fat diet had roughly a 25% shorter life span compared with control animals. This study highlights the potential of ART to induce vascular dysfunction and shorten life span and suggests that epigenetic alterations contribute to these problems.

Fetal programming of pulmonary vascular dysfunction in mice: role of epigenetic mechanisms.

Insults during the fetal period predispose the offspring to systemic cardiovascular disease, but little is known about the pulmonary circulation and the underlying mechanisms. Maternal undernutrition during pregnancy may represent a model to investigate underlying mechanisms, because it is associated with systemic vascular dysfunction in the offspring in animals and humans. In rats, restrictive diet during pregnancy (RDP) increases oxidative stress in the placenta. Oxygen species are known to induce epigenetic alterations and may cross the placental barrier. We hypothesized that RDP in mice induces pulmonary vascular dysfunction in the offspring that is related to an epigenetic mechanism. To test this hypothesis, we assessed pulmonary vascular function and lung DNA methylation in offspring of RDP and in control mice at the end of a 2-wk exposure to hypoxia. We found that endothelium-dependent pulmonary artery vasodilation in vitro was impaired and hypoxia-induced pulmonary hypertension and right ventricular hypertrophy in vivo were exaggerated in offspring of RDP. This pulmonary vascular dysfunction was associated with altered lung DNA methylation. Administration of the histone deacetylase inhibitors butyrate and trichostatin A to offspring of RDP normalized pulmonary DNA methylation and vascular function. Finally, administration of the nitroxide Tempol to the mother during RDP prevented vascular dysfunction and dysmethylation in the offspring. These findings demonstrate that in mice undernutrition during gestation induces pulmonary vascular dysfunction in the offspring by an epigenetic mechanism. A similar mechanism may be involved in the fetal programming of vascular dysfunction in humans.

A BAYESIAN APPROACH TO DOSE-RESPONSE ASSESSMENT AND DRUG-DRUG INTERACTION ANALYSIS: APPLICATION TO IN VITRO STUDIES

The considerable search for synergistic agents in cancer research is motivated by the therapeutic benefits achieved by combining anti-cancer agents. Synergistic agents make it possible to reduce dosage while maintaining or enhancing a desired effect. Other favorable outcomes of synergistic agents include reduction in toxicity and minimizing or delaying drug resistance. Dose-response assessment and drug-drug interaction analysis play an important part in the drug discovery process, however analysis are often poorly done. This dissertation is an effort to notably improve dose-response assessment and drug-drug interaction analysis. The most commonly used method in published analysis is the Median-Effect Principle/Combination Index method (Chou and Talalay, 1984). The Median-Effect Principle/Combination Index method leads to inefficiency by ignoring important sources of variation inherent in dose-response data and discarding data points that do not fit the Median-Effect Principle. Previous work has shown that the conventional method yields a high rate of false positives (Boik, Boik, Newman, 2008; Hennessey, Rosner, Bast, Chen, 2010) and, in some cases, low power to detect synergy. There is a great need for improving the current methodology. We developed a Bayesian framework for dose-response modeling and drug-drug interaction analysis. First, we developed a hierarchical meta-regression dose-response model that accounts for various sources of variation and uncertainty and allows one to incorporate knowledge from prior studies into the current analysis, thus offering a more efficient and reliable inference. Second, in the case that parametric dose-response models do not fit the data, we developed a practical and flexible nonparametric regression method for meta-analysis of independently repeated dose-response experiments. Third, and lastly, we developed a method, based on Loewe additivity that allows one to quantitatively assess interaction between two agents combined at a fixed dose ratio. The proposed method makes a comprehensive and honest account of uncertainty within drug interaction assessment. Extensive simulation studies show that the novel methodology improves the screening process of effective/synergistic agents and reduces the incidence of type I error. We consider an ovarian cancer cell line study that investigates the combined effect of DNA methylation inhibitors and histone deacetylation inhibitors in human ovarian cancer cell lines. The hypothesis is that the combination of DNA methylation inhibitors and histone deacetylation inhibitors will enhance antiproliferative activity in human ovarian cancer cell lines compared to treatment with each inhibitor alone. By applying the proposed Bayesian methodology, in vitro synergy was declared for DNA methylation inhibitor, 5-AZA-2'-deoxycytidine combined with one histone deacetylation inhibitor, suberoylanilide hydroxamic acid or trichostatin A in the cell lines HEY and SKOV3. This suggests potential new epigenetic therapies in cell growth inhibition of ovarian cancer cells.

Identification of a SIRT1 mutation in a family with type 1 diabetes.

Type 1 diabetes is caused by autoimmune-mediated β cell destruction leading to insulin deficiency. The histone deacetylase SIRT1 plays an essential role in modulating several age-related diseases. Here we describe a family carrying a mutation in the SIRT1 gene, in which all five affected members developed an autoimmune disorder: four developed type 1 diabetes, and one developed ulcerative colitis. Initially, a 26-year-old man was diagnosed with the typical features of type 1 diabetes, including lean body mass, autoantibodies, T cell reactivity to β cell antigens, and a rapid dependence on insulin. Direct and exome sequencing identified the presence of a T-to-C exchange in exon 1 of SIRT1, corresponding to a leucine-to-proline mutation at residue 107. Expression of SIRT1-L107P in insulin-producing cells resulted in overproduction of nitric oxide, cytokines, and chemokines. These observations identify a role for SIRT1 in human autoimmunity and unveil a monogenic form of type 1 diabetes.

ROLE OF SYNAPSIN IN LONG-TERM SYNAPTIC FACILITATION IN APLYSIA

Enhanced expression of the presynaptic protein synapsin has been correlated with certain forms of long-term plasticity and learning and memory. However, the regulation and requirement for enhanced synapsin expression in long-term memory remains unknown. In the present study the technical advantages of the marine mollusc Aplysia were exploited in order to address this issue. In Aplysia, learning-induced enhancement in synaptic strength is modulated by serotonin (5-HT) and treatment with 5-HT in vitro of the sensorimotor synapse induces long-term facilitation (LTF) of synaptic transmission, which lasts for days, as well as the formation of new connections between the sensory and motor neuron. Results from immunofluorescence analysis indicated that 5-HT treatment upregulates synapsin protein levels within sensory neuron varicosities, the presumed site of neurotransmitter release. To investigate the mechanisms underlying increased synapsin expression, the promoter region of the Aplysia synapsin gene was cloned and a cAMP response element (CRE) was identified, raising the possibility that the transcriptional activator cAMP response element-binding protein-1 (CREB1) mediates the 5-HT-induced regulation of synapsin. Results from Chromatin Immunoprecipitation (ChIP) assays indicated that 5-HT treatment enhanced association of CREB1 surrounding the CRE site in the synapsin promoter and led to increased acetylation of histones H3 and H4 and decreased association of histone deacetylase 5 surrounding the CRE site in the synapsin promoter, a sign of transcriptional activation. In addition, sensory neurons injected with an enhanced green fluorescent protein (EGFP) reporter vector driven by the synapsin promoter exhibited a significant increase in EGFP expression following treatment with 5-HT. These results suggest that synapsin expression is regulated by 5-HT in part through transcriptional activation of the synapsin gene and through CREB1 association with the synapsin promoter. Furthermore, RNA interference that blocks 5-HT-induced elevation of synapsin expression also blocked long-term synaptic facilitation. These results indicate that 5-HT-induced regulation of synapsin is necessary for LTF and that synapsin is part of the cascade of synaptic events involved in the consolidation of memory.

DEFINITION OF THE LANDSCAPE OF CHROMATIN STRUCTURE AT THE FRATAXIN GENE IN FRIEDREICH’S ATAXIA

Friedreich’s ataxia (FRDA) is caused by the transcriptional silencing of the frataxin (FXN) gene. FRDA patients have expansion of GAA repeats in intron 1 of the FXN gene in both alleles. A number of studies demonstrated that specific histone deacetylase inhibitors (HDACi) affect either histone modifications at the FXN gene or FXN expression in FRDA cells, indicating that the hyperexpanded GAA repeat may facilitate heterochromatin formation. However, the correlation between chromatin structure and transcription at the FXN gene is currently limited due to a lack of more detailed analysis. Therefore, I analyzed the effects of the hyperexpanded GAA repeats on transcription status and chromatin structure using lymphoid cell lines derived from FRDA patients. Using chromatin immunoprecipitation and quantitative PCR, I observed significant changes in the landscape of histone modifications in the vicinity of the GAA tract in FRDA cells relative to control cells. Similar epigenetic changes were observed in GFP reporter construct containing 560 GAA repeats. Further, I detected similar levels of FXN pre-mRNA at a region upstream of hyperexpanded GAA repeats in FRDA and control cells, indicating similar efficiency of transcription initiation in FRDA cells. I also showed that histone modifications associated with hyperexpanded GAA repeats are independent of transcription progression using the GFP reporter system. My data strongly support evidence that FXN deficiency in FRDA patients is consequence of defective transition from initiation to elongation of FXN transcription due to heterochromatin-like structures formed in the proximity of the hyperexpanded GAAs.

Loss of Sirt1 function improves intestinal anti-bacterial defense and protects from colitis-induced colorectal cancer

Dysfunction of Paneth and goblet cells in the intestine contributes to inflammatory bowel disease (IBD) and colitis-associated colorectal cancer (CAC). Here, we report a role for the NAD+-dependent histone deacetylase SIRT1 in the control of anti-bacterial defense. Mice with an intestinal specific Sirt1 deficiency (Sirt1int-/-) have more Paneth and goblet cells with a consequent rearrangement of the gut microbiota. From a mechanistic point of view, the effects on mouse intestinal cell maturation are mediated by SIRT1-dependent changes in the acetylation status of SPDEF, a master regulator of Paneth and goblet cells. Our results suggest that targeting SIRT1 may be of interest in the management of IBD and CAC.

p21WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells

A diet high in fiber is associated with a decreased incidence and growth of colon cancers. Butyrate, a four-carbon short-chain fatty acid product of fiber fermentation within the colon, appears to mediate these salutary effects. We sought to determine the molecular mechanism by which butyrate mediates growth inhibition of colonic cancer cells and thereby to elucidate the molecular link between a high-fiber diet and the arrest of colon carcinogenesis. We show that concomitant with growth arrest, butyrate induces p21 mRNA expression in an immediate-early fashion, through transactivation of a promoter cis-element(s) located within 1.4 kb of the transcriptional start site, independent of p53 binding. Studies using the specific histone hyperacetylating agent, trichostatin A, and histone deacetylase 1 indicate that growth arrest and p21 induction occur through a mechanism involving histone hyperacetylation. We show the critical importance of p21 in butyrate-mediated growth arrest by first confirming that stable overexpression of the p21 gene is able to cause growth arrest in the human colon carcinoma cell line, HT-29. Furthermore, using p21-deleted HCT116 human colon carcinoma cells, we provide convincing evidence that p21 is required for growth arrest to occur in response to histone hyperacetylation, but not for serum starvation nor postconfluent growth. Thus, p21 appears to be a critical effector of butyrate-induced growth arrest in colonic cancer cells, and may be an important molecular link between a high-fiber diet and the prevention of colon carcinogenesis.

Crystal structure of the BTB domain from PLZF

The BTB domain (also known as the POZ domain) is an evolutionarily conserved protein–protein interaction motif found at the N terminus of 5–10% of C2H2-type zinc-finger transcription factors, as well as in some actin-associated proteins bearing the kelch motif. Many BTB proteins are transcriptional regulators that mediate gene expression through the control of chromatin conformation. In the human promyelocytic leukemia zinc finger (PLZF) protein, the BTB domain has transcriptional repression activity, directs the protein to a nuclear punctate pattern, and interacts with components of the histone deacetylase complex. The association of the PLZF BTB domain with the histone deacetylase complex provides a mechanism of linking the transcription factor with enzymatic activities that regulate chromatin conformation. The crystal structure of the BTB domain of PLZF was determined at 1.9 Å resolution and reveals a tightly intertwined dimer with an extensive hydrophobic interface. Approximately one-quarter of the monomer surface area is involved in the dimer intermolecular contact. These features are typical of obligate homodimers, and we expect the full-length PLZF protein to exist as a branched transcription factor with two C-terminal DNA-binding regions. A surface-exposed groove lined with conserved amino acids is formed at the dimer interface, suggestive of a peptide-binding site. This groove may represent the site of interaction of the PLZF BTB domain with nuclear corepressors or other nuclear proteins.

Corepressor SMRT binds the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein

The LAZ3/BCL6 (lymphoma-associated zinc finger 3/B cell lymphomas 6) gene frequently is altered in non-Hodgkin lymphomas. It encodes a sequence-specific DNA binding transcriptional repressor that contains a conserved N-terminal domain, termed BTB/POZ (bric-à-brac tramtrack broad complex/pox viruses and zinc fingers). Using a yeast two-hybrid screen, we show here that the LAZ3/BCL6 BTB/POZ domain interacts with the SMRT (silencing mediator of retinoid and thyroid receptor) protein. SMRT originally was identified as a corepressor of unliganded retinoic acid and thyroid receptors and forms a repressive complex with a mammalian homolog of the yeast transcriptional repressor SIN3 and the HDAC-1 histone deacetylase. Protein binding assays demonstrate that the LAZ3/BCL6 BTB/POZ domain directly interacts with SMRT in vitro. Furthermore, DNA-bound LAZ3/BCL6 recruits SMRT in vivo, and both overexpressed proteins completely colocalize in nuclear dots. Finally, overexpression of SMRT enhances the LAZ3/BCL6-mediated repression. These results define SMRT as a corepressor of LAZ3/BCL6 and suggest that LAZ3/BCL6 and nuclear hormone receptors repress transcription through shared mechanisms involving SMRT recruitment and histone deacetylation.

ETO, fusion partner in t(8;21) acute myeloid leukemia, represses transcription by interaction with the human N-CoR/mSin3/HDAC1 complex

The t(8;21) translocation between two genes known as AML1 and ETO is seen in approximately 12–15% of all acute myeloid leukemia (AML) and is the second-most-frequently observed nonrandom genetic alteration associated with AML. AML1 up-regulates a number of target genes critical to normal hematopoiesis, whereas the AML1/ETO fusion interferes with this trans-activation. We discovered that the fusion partner ETO binds to the human homolog of the murine nuclear receptor corepressor (N-CoR). The interaction is mediated by two unusual zinc finger motifs present at the carboxyl terminus of ETO. Human N-CoR (HuN-CoR), which we cloned and sequenced in its entirety, encodes a 2,440-amino acid polypeptide and has a central domain that binds ETO. N-CoR, mammalian Sin3 (mSin3A and B), and histone deacetylase 1 (HDAC1) form a complex that alters chromatin structure and mediates transcriptional repression by nuclear receptors and by a number of oncoregulatory proteins. We found that ETO, through its interaction with the N-CoR/mSin3/HDAC1 complex, is also a potent repressor of transcription. This observation provides a mechanism for how the AML1/ETO fusion may inhibit expression of AML1-responsive target genes and disturb normal hematopoiesis.

All Tcf HMG box transcription factors interact with Groucho-related co-repressors

Tcf/Lef family transcription factors are the downstream effectors of the Wingless/Wnt signal transduction pathway. Upon Wingless/Wnt signalling, β-catenin translocates to the nucleus, interacts with Tcf (1–3) and thus activates transcription of target genes (4,5). Tcf factors also interact with members of the Groucho (Grg/TLE) family of transcriptional co-repressors (6). We have now tested all known mammalian Groucho family members for their ability to interact specifically with individual Tcf/Lef family members. Transcriptional activation by any Tcf could be repressed by Grg-1, Grg-2/TLE-2, Grg-3 and Grg-4 in a reporter assay. Specific interactions between Tcf and Grg proteins may be achieved in vivo by tissue- or cell type-limited expression. To address this, we determined the expression of all Tcf and Grg/TLE family members in a panel of cell lines. Within any cell line, several Tcfs and TLEs are co-expressed. Thus, redundancy in Tcf/Grg interactions appears to be the rule. The ‘long’ Groucho family members containing five domains are repressors of Tcf-mediated transactivation, whereas Grg-5, which only contains the first two domains, acts as a de-repressor. As previously shown for Drosophila Groucho, we show that long Grg proteins interact with histone deacetylase-1. Although Grg-5 contains the GP homology domain that mediates HDAC binding in long Grg proteins, Grg-5 fails to bind this co-repressor, explaining how it can de-repress transcription.

Difficult macrocyclizations: New strategies for synthesizing highly strained cyclic tetrapeptides

Cyclic tetrapeptides are an intriguing class of natural products. To synthesize highly strained cyclic tetrapeptides; we developed a macrocyclization strategy that involves the inclusion of 2-hydroxy-6-nitrobenzyl (HnB) group at the N-terminus and in the middle of the sequence. The N-terminal auxiliary performs a ring closure/ring contraction role, and the backbone auxiliary promotes cis amide bonds to facilitate the otherwise difficult ring contraction. Following this route, the all-L cyclic tetrapeptide cyclo-[Tyr-Arg-Phe-Ala] was successfully prepared.