Publications by authors named "Gianluca Fossati"

31 Publications

Epigenetic-based cancer therapeutics: new potential HDAC8 inhibitors.

J Biomol Struct Dyn 2020 Sep 4:1-15. Epub 2020 Sep 4.

Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.

Designing dual small molecule inhibitors against enzymes associated with cancer has turned into a new strategy in cancer chemotherapy. Targeting DNA methyltransferase (DNMT) and histone deacetylase (HDAC) enzymes, involved in epigenetic modifications, are considered as promising treatments for a wide range of cancers, due to their association with the initiation, proliferation, and survival of cancer cells. In this study, for the first time, the dual inhibitors of the histone deacetylases 8 (HDAC8) and DNA methyltransferase 1 (DNMT1) has introduced as novel potential candidates for epigenetic-based cancer therapeutics. This research has been facilitated by employing pharmacophore-based virtual screening of ZINC and Maybridge databases, as well as performing molecular docking, molecular dynamics simulations and free binding energy calculation on the top derived compound. Results have demonstrated that the suggested compounds not only adopt highly favorable conformations but also possess strong binding interaction with the HDAC8 enzyme. Additionally, the obtained results from the experimental assay confirmed the predicted behavior of inhibitors from virtual screening. These results can be used for further optimization to yield promising more effective candidates for the treatment of cancer.Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2020.1813203DOI Listing
September 2020

Novel Benzohydroxamate-Based Potent and Selective Histone Deacetylase 6 (HDAC6) Inhibitors Bearing a Pentaheterocyclic Scaffold: Design, Synthesis, and Biological Evaluation.

J Med Chem 2019 12 25;62(23):10711-10739. Epub 2019 Nov 25.

Preclinical R&D , Italfarmaco Group , Via dei Lavoratori 54 , I-20092 Cinisello Balsamo , Milan , Italy.

Histone deacetylase 6 (HDAC6) is a peculiar HDAC isoform whose expression and functional alterations have been correlated with a variety of pathologies such as autoimmune disorders, neurodegenerative diseases, and cancer. It is primarily a cytoplasmic protein, and its deacetylase activity is focused mainly on nonhistone substrates such as tubulin, heat shock protein (HSP)90, Foxp3, and cortactin, to name a few. Selective inhibition of HDAC6 does not show cytotoxic effects in healthy cells, normally associated with the inhibition of Class I HDAC isoforms. Here, we describe the design and synthesis of a new class of potent and selective HDAC6 inhibitors that bear a pentaheterocyclic central core. These compounds show a remarkably low toxicity both in vitro and in vivo and are able to increase the function of regulatory T cells (Tregs) at well-tolerated concentrations, suggesting a potential clinical use for the treatment of degenerative, autoimmune diseases and for organ transplantation.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01194DOI Listing
December 2019

Improved In Vivo Anti-Tumor and Anti-Metastatic Effect of GnRH-III-Daunorubicin Analogs on Colorectal and Breast Carcinoma Bearing Mice.

Int J Mol Sci 2019 Sep 25;20(19). Epub 2019 Sep 25.

Department of Experimental Pharmacology, National Institute of Oncology, 1122 Budapest, Hungary.

Among various homing devices, gonadotropin-releasing hormone-III (GnRH-III) peptide represents a suitable targeting moiety for drug delivery systems. The anti-tumor activity of the previously developed GnRH-III-[Lys(Bu),Lys(Dau=Aoa)] conjugate and the novel synthesized GnRH-III-[ΔHis,d-Tic,Lys(Bu),Lys(Dau=Aoa)] conjugate, containing the anti-cancer drug daunorubicin, were evaluated. Here, we demonstrate that both GnRH-III-Dau conjugates possess an efficient growth inhibitory effect on more than 20 cancer cell lines, whereby the biological activity is strongly connected to the expression of gonadotropin-releasing hormone receptors (GnRH-R). The novel conjugate showed a higher in vitro anti-proliferative activity and a higher uptake capacity. Moreover, the treatment with GnRH-III-Dau conjugates cause a significant in vivo tumor growth and metastases inhibitory effect in three different orthotopic models, including 4T1 mice and MDA-MB-231 human breast carcinoma, as well as HT-29 human colorectal cancer bearing BALB/s and SCID mice, while toxic side-effects were substantially reduced in comparison to the treatment with the free drug. These findings illustrate that our novel lead compound is a highly promising candidate for targeted tumor therapy in both colon cancer and metastatic breast cancer.
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http://dx.doi.org/10.3390/ijms20194763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801585PMC
September 2019

Engineered hexavalent Fc proteins with enhanced Fc-gamma receptor avidity provide insights into immune-complex interactions.

Commun Biol 2018 14;1:146. Epub 2018 Sep 14.

UCB Pharma, 216 Bath Road, Slough, SL1 3WE, UK.

Autoantibody-mediated diseases are currently treated with intravenous immunoglobulin, which is thought to act in part via blockade of Fc gamma receptors, thereby inhibiting autoantibody effector functions and subsequent pathology. We aimed to develop recombinant molecules with enhanced Fc receptor avidity and thus increased potency over intravenous immunoglobulin. Here we describe the molecular engineering of human Fc hexamers and explore their therapeutic and safety profiles. We show Fc hexamers were more potent than IVIG in phagocytosis blockade and disease models. However, in human whole-blood safety assays incubation with IgG1 isotype Fc hexamers resulted in cytokine release, platelet and complement activation, whereas the IgG4 version did not. We used a statistically designed mutagenesis approach to identify the key Fc residues involved in these processes. Cytokine release was found to be dependent on neutrophil FcγRIIIb interactions with L234 and A327 in the Fc. Therefore, Fc hexamers provide unique insights into Fc receptor biology.
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http://dx.doi.org/10.1038/s42003-018-0149-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138732PMC
September 2018

Control of cytokine mRNA degradation by the histone deacetylase inhibitor ITF2357 in rheumatoid arthritis fibroblast-like synoviocytes: beyond transcriptional regulation.

Arthritis Res Ther 2018 07 20;20(1):148. Epub 2018 Jul 20.

Laboratory of Translational Immunology and Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: Histone deacetylase inhibitors (HDACi) suppress cytokine production in immune and stromal cells of patients with rheumatoid arthritis (RA). Here, we investigated the effects of the HDACi givinostat (ITF2357) on the transcriptional and post-transcriptional regulation of inflammatory markers in RA fibroblast-like synoviocytes (FLS).

Methods: The effects of ITF2357 on the expression and messenger RNA (mRNA) stability of IL-1β-inducible genes in FLS were analyzed using array-based qPCR and Luminex. The expression of primary and mature cytokine transcripts, the mRNA levels of tristetraprolin (TTP, or ZFP36) and other AU-rich element binding proteins (ARE-BP) and the cytokine profile of fibroblasts derived from ZFP36 and ZFP36 mice was measured by qPCR. ARE-BP silencing was performed by small interfering RNA (siRNA)-mediated knockdown, and TTP post-translational modifications were analyzed by immunoblotting.

Results: ITF2357 reduced the expression of 85% of the analyzed IL-1β-inducible transcripts, including cytokines (IL6, IL8), chemokines (CXCL2, CXCL5, CXCL6, CXCL10), matrix-degrading enzymes (MMP1, ADAMTS1) and other inflammatory mediators. Analyses of mRNA stability demonstrated that ITF2357 accelerates IL6, IL8, PTGS2 and CXCL2 mRNA degradation, a phenomenon associated with the enhanced transcription of TTP, but not other ARE-BP, and the altered post-translational status of TTP protein. TTP knockdown potentiated cytokine production in RA FLS and murine fibroblasts, which in the latter case was insensitive to inhibition by ITF2357 treatment.

Conclusions: Our study identifies that regulation of cytokine mRNA stability is a predominant mechanism underlying ITF2357 anti-inflammatory properties, occurring via regulation of TTP. These results highlight the therapeutic potential of ITF2357 in the treatment of RA.
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http://dx.doi.org/10.1186/s13075-018-1638-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053802PMC
July 2018

Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells.

Oncogene 2018 07 12;37(28):3839-3851. Epub 2018 Apr 12.

Istituto Oncologico Veneto IOV-IRCCS, Padova, Italy.

Several studies have revealed that endosomal sorting controls the steady-state levels of Notch at the cell surface in normal cells and prevents its inappropriate activation in the absence of ligands. However, whether this highly dynamic physiologic process can be exploited to counteract dysregulated Notch signaling in cancer cells remains unknown. T-ALL is a malignancy characterized by aberrant Notch signaling, sustained by activating mutations in Notch1 as well as overexpression of Notch3, a Notch paralog physiologically subjected to lysosome-dependent degradation in human cancer cells. Here we show that treatment with the pan-HDAC inhibitor Trichostatin A (TSA) strongly decreases Notch3 full-length protein levels in T-ALL cell lines and primary human T-ALL cells xenografted in mice without substantially reducing NOTCH3 mRNA levels. Moreover, TSA markedly reduced the levels of Notch target genes, including pTα, CR2, and DTX-1, and induced apoptosis of T-ALL cells. We further observed that Notch3 was post-translationally regulated following TSA treatment, with reduced Notch3 surface levels and increased accumulation of Notch3 protein in the lysosomal compartment. Surface Notch3 levels were rescued by inhibition of dynein with ciliobrevin D. Pharmacologic studies with HDAC1, 6, and 8-specific inhibitors disclosed that these effects were largely due to inhibition of HDAC6 in T-ALL cells. HDAC6 silencing by specific shRNA was followed by reduced Notch3 expression and increased apoptosis of T-ALL cells. Finally, HDAC6 silencing impaired leukemia outgrowth in mice, associated with reduction of Notch3 full-length protein in vivo. These results connect HDAC6 activity to regulation of total and surface Notch3 levels and suggest HDAC6 as a potential novel therapeutic target to lower Notch signaling in T-ALL and other Notch3-addicted tumors.
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http://dx.doi.org/10.1038/s41388-018-0234-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6041259PMC
July 2018

Inhibition of the Autophagy Pathway Synergistically Potentiates the Cytotoxic Activity of Givinostat (ITF2357) on Human Glioblastoma Cancer Stem Cells.

Front Mol Neurosci 2016 27;9:107. Epub 2016 Oct 27.

Department of Biology and Biotechnology, University of Pavia Pavia, Italy.

Increasing evidence highlighted the role of cancer stem cells (CSCs) in the development of tumor resistance to therapy, particularly in glioblastoma (GBM). Therefore, the development of new therapies, specifically directed against GBM CSCs, constitutes an important research avenue. Considering the extended range of cancer-related pathways modulated by histone acetylation/deacetylation processes, we studied the anti-proliferative and pro-apoptotic efficacy of givinostat (GVS), a pan-histone deacetylase inhibitor, on cell cultures enriched in CSCs, isolated from nine human GBMs. We report that GVS induced a significant reduction of viability and self-renewal ability in all GBM CSC cultures; conversely, GVS exposure did not cause a significant cytotoxic activity toward differentiated GBM cells and normal mesenchymal human stem cells. Analyzing the cellular and molecular mechanisms involved, we demonstrated that GVS affected CSC viability through the activation of programmed cell death pathways. In particular, a marked stimulation of macroautophagy was observed after GVS treatment. To understand the functional link between GVS treatment and autophagy activation, different genetic and pharmacological interfering strategies were used. We show that the up-regulation of the autophagy process, obtained by deprivation of growth factors, induced a reduction of CSC sensitivity to GVS, while the pharmacological inhibition of the autophagy pathway and the silencing of the key autophagy gene , increased the cell death rate induced by GVS. Altogether these findings suggest that autophagy represents a pro-survival mechanism activated by GBM CSCs to counteract the efficacy of the anti-proliferative activity of GVS. In conclusion, we demonstrate that GVS is a novel pharmacological tool able to target GBM CSC viability and its efficacy can be enhanced by autophagy inhibitory strategies.
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http://dx.doi.org/10.3389/fnmol.2016.00107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081386PMC
October 2016

Histone deacetylase 3 regulates the inflammatory gene expression programme of rheumatoid arthritis fibroblast-like synoviocytes.

Ann Rheum Dis 2017 Jan 25;76(1):277-285. Epub 2016 Jul 25.

Department of Experimental Immunology and Department of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Objectives: Non-selective histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated anti-inflammatory properties in both in vitro and in vivo models of rheumatoid arthritis (RA). Here, we investigated the potential contribution of specific class I and class IIb HDACs to inflammatory gene expression in RA fibroblast-like synoviocytes (FLS).

Methods: RA FLS were incubated with pan-HDACi (ITF2357, givinostat) or selective HDAC1/2i, HDAC3/6i, HDAC6i and HDAC8i. Alternatively, FLS were transfected with HDAC3, HDAC6 or interferon (IFN)-α/β receptor alpha chain (IFNAR1) siRNA. mRNA expression of interleukin (IL)-1β-inducible genes was measured by quantitative PCR (qPCR) array and signalling pathway activation by immunoblotting and DNA-binding assays.

Results: HDAC3/6i, but not HDAC1/2i and HDAC8i, significantly suppressed the majority of IL-1β-inducible genes targeted by pan-HDACi in RA FLS. Silencing of HDAC3 expression reproduced the effects of HDAC3/6i on gene regulation, contrary to HDAC6-specific inhibition and HDAC6 silencing. Screening of the candidate signal transducers and activators of transcription (STAT)1 transcription factor revealed that HDAC3/6i abrogated STAT1 Tyr701 phosphorylation and DNA binding, but did not affect STAT1 acetylation. HDAC3 activity was required for type I IFN production and subsequent STAT1 activation in FLS. Suppression of type I IFN release by HDAC3/6i resulted in reduced expression of a subset of IFN-dependent genes, including the chemokines CXCL9 and CXCL11.

Conclusions: Inhibition of HDAC3 in RA FLS largely recapitulates the effects of pan-HDACi in suppressing inflammatory gene expression, including type I IFN production in RA FLS. Our results identify HDAC3 as a potential therapeutic target in the treatment of RA and type I IFN-driven autoimmune diseases.
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http://dx.doi.org/10.1136/annrheumdis-2015-209064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264225PMC
January 2017

Novel HDAC inhibitors exhibit pre-clinical efficacy in lymphoma models and point to the importance of CDKN1A expression levels in mediating their anti-tumor response.

Oncotarget 2015 Mar;6(7):5059-71

Lymphoma & Genomics Research Program, IOR Institute of Oncology Research, Bellinzona, Switzerland.

We investigated the pre-clinical activities of two novel histone deacetylase inhibitors (HDACi), ITF-A and ITF-B, in a large panel of pre-clinical lymphoma models. The two compounds showed a dose-dependent anti-proliferative activity in the majority of cell lines. Gene expression profiling (GEP) of diffuse large B-cell lymphoma (DLBCL) cells treated with the compounds showed a modulation of genes involved in chromatin structure, cell cycle progression, apoptosis, B-cell signaling, and genes encoding metallothioneins. Cell lines showed differences between the concentrations of ITF-A and ITF-B needed to cause anti-proliferative or cytotoxic activity, and cell cycle and apoptosis genes appeared implicated in determining the type of response. In particular, CDKN1A expression was higher in DLBCL cells that, to undergo apoptosis, required a much higher amount of drug than that necessary to induce only an anti-proliferative effect.In conclusion, the two novel HDACi ITF-A and ITF-B demonstrated anti-proliferative activity across different mature B-cell lymphoma cell lines. Basal CDKN1A levels appeared to be important in determining the gap between HDACi concentrations causing cell cycle arrest and those that lead to cell death.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4467133PMC
http://dx.doi.org/10.18632/oncotarget.3239DOI Listing
March 2015

Suppression of monosodium urate crystal-induced cytokine production by butyrate is mediated by the inhibition of class I histone deacetylases.

Ann Rheum Dis 2016 Mar 14;75(3):593-600. Epub 2015 Jan 14.

Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.

Objectives: Acute gouty arthritis is caused by endogenously formed monosodium urate (MSU) crystals, which are potent activators of the NLRP3 inflammasome. However, to induce the release of active interleukin (IL)-1β, an additional stimulus is needed. Saturated long-chain free fatty acids (FFAs) can provide such a signal and stimulate transcription of pro-IL-1β. In contrast, the short-chain fatty acid butyrate possesses anti-inflammatory effects. One of the mechanisms involved is inhibition of histone deacetylases (HDACs). Here, we explored the effects of butyrate on MSU+FFA-induced cytokine production and its inhibition of specific HDACs.

Methods: Freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with MSU and palmitic acid (C16.0) in the presence or absence of butyrate or a synthetic HDAC inhibitor. Cytokine responses were measured with ELISA and quantitative PCR. HDAC activity was measured with fluorimetric assays.

Results: Butyrate decreased C16.0+MSU-induced production of IL-1β, IL-6, IL-8 and IL-1β mRNA in PBMCs from healthy donors. Similar results were obtained in PBMCs isolated from patients with gout. Butyrate specifically inhibited class I HDACs. The HDAC inhibitor, panobinostat and the potent HDAC inhibitor, ITF-B, also decreased ex vivo C16.0+MSU-induced IL-1β production.

Conclusions: In agreement with the reported low inhibitory potency of butyrate, a high concentration was needed for cytokine suppression, whereas synthetic HDAC inhibitors showed potent anti-inflammatory effects at nanomolar concentrations. These novel HDAC inhibitors could be effective in the treatment of acute gout. Moreover, the use of specific HDAC inhibitors could even improve the efficacy and reduce any potential adverse effects.
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http://dx.doi.org/10.1136/annrheumdis-2014-206258DOI Listing
March 2016

Specific inhibition of histone deacetylase 8 reduces gene expression and production of proinflammatory cytokines in vitro and in vivo.

J Biol Chem 2015 Jan 1;290(4):2368-78. Epub 2014 Dec 1.

From the Department of Medicine, University of Colorado Denver, Aurora, Colorado 80045, the Department of Medicine, Radboud University Medical Centre, 6525 HP Nijmegen, The Netherlands

ITF2357 (generic givinostat) is an orally active, hydroxamic-containing histone deacetylase (HDAC) inhibitor with broad anti-inflammatory properties, which has been used to treat children with systemic juvenile idiopathic arthritis. ITF2357 inhibits both Class I and II HDACs and reduces caspase-1 activity in human peripheral blood mononuclear cells and the secretion of IL-1β and other cytokines at 25-100 nm; at concentrations >200 nm, ITF2357 is toxic in vitro. ITF3056, an analog of ITF2357, inhibits only HDAC8 (IC50 of 285 nm). Here we compared the production of IL-1β, IL-1α, TNFα, and IL-6 by ITF2357 with that of ITF3056 in peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), heat-killed Candida albicans, or anti-CD3/anti-CD28 antibodies. ITF3056 reduced LPS-induced cytokines from 100 to 1000 nm; at 1000 nm, the secretion of IL-1β was reduced by 76%, secretion of TNFα was reduced by 88%, and secretion of IL-6 was reduced by 61%. The intracellular levels of IL-1α were 30% lower. There was no evidence of cell toxicity at ITF3056 concentrations of 100-1000 nm. Gene expression of TNFα was markedly reduced (80%), whereas IL-6 gene expression was 40% lower. Although anti-CD3/28 and Candida stimulation of IL-1β and TNFα was modestly reduced, IFNγ production was 75% lower. Mechanistically, ITF3056 reduced the secretion of processed IL-1β independent of inhibition of caspase-1 activity; however, synthesis of the IL-1β precursor was reduced by 40% without significant decrease in IL-1β mRNA levels. In mice, ITF3056 reduced LPS-induced serum TNFα by 85% and reduced IL-1β by 88%. These data suggest that specific inhibition of HDAC8 results in reduced inflammation without cell toxicity.
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http://dx.doi.org/10.1074/jbc.M114.618454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303687PMC
January 2015

Modulation of gamma globin genes expression by histone deacetylase inhibitors: an in vitro study.

Br J Haematol 2014 Jun 7;165(5):714-21. Epub 2014 Mar 7.

Department of Clinical Sciences and Community, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy; UO Genetica Medica, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy.

Induction of fetal haemoglobin (HbF) is a promising therapeutic approach for the treatment of β-thalassaemia and sickle cell disease (SCD). Several pharmacological agents, such as hydroxycarbamide (HC) and butyrates, have been shown to induce the γ-globin genes (HBG1, HBG2). However, their therapeutic use is limited due to weak efficacy and an inhibitory effect on erythroid differentiation. Thus, more effective agents are needed. The histone deacetylase (HDAC) inhibitors are potential therapeutic haemoglobin (Hb) inducers able to modulate gene expression through pleiotropic mechanisms. We investigated the effects of a HDAC inhibitor, Givinostat (GVS), on erythropoiesis and haemoglobin synthesis and compared it with sodium butyrate and HC. We used an in vitro erythropoiesis model derived from peripheral CD34⁺ cells of healthy volunteers and SCD donors. GVS effects on erythroid proliferation and differentiation and on Hb synthesis were investigated. We found that GVS at high concentrations delayed erythroid differentiation with no specific effect on HBG1/2 transcription. At a low concentration (1 nmol/l), GVS induced Hb production with no effects on cells proliferation and differentiation. The efficacy of GVS 1 mol/l in Hb induction in vitro was comparable to that of HC and butyrate. Our results support the evaluation of GVS as a new candidate molecule for the treatment of the haemoglobinophathies due to its positive effects on haemoglobin production at low and non-toxic concentrations.
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http://dx.doi.org/10.1111/bjh.12814DOI Listing
June 2014

Lysine deacetylase inhibition prevents diabetes by chromatin-independent immunoregulation and β-cell protection.

Proc Natl Acad Sci U S A 2014 Jan 6;111(3):1055-9. Epub 2014 Jan 6.

Section for Endocrinological Research, Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.

Type 1 diabetes is due to destruction of pancreatic β-cells. Lysine deacetylase inhibitors (KDACi) protect β-cells from inflammatory destruction in vitro and are promising immunomodulators. Here we demonstrate that the clinically well-tolerated KDACi vorinostat and givinostat revert diabetes in the nonobese diabetic (NOD) mouse model of type 1 diabetes and counteract inflammatory target cell damage by a mechanism of action consistent with transcription factor--rather than global chromatin--hyperacetylation. Weaning NOD mice received low doses of vorinostat and givinostat in their drinking water until 100-120 d of age. Diabetes incidence was reduced by 38% and 45%, respectively, there was a 15% increase in the percentage of islets without infiltration, and pancreatic insulin content increased by 200%. Vorinostat treatment increased the frequency of functional regulatory T-cell subsets and their transcription factors Gata3 and FoxP3 in parallel to a decrease in inflammatory dendritic cell subsets and their cytokines IL-6, IL-12, and TNF-α. KDACi also inhibited LPS-induced Cox-2 expression in peritoneal macrophages from C57BL/6 and NOD mice. In insulin-producing β-cells, givinostat did not upregulate expression of the anti-inflammatory genes Socs1-3 or sirtuin-1 but reduced levels of IL-1β + IFN-γ-induced proinflammatory Il1a, Il1b, Tnfα, Fas, Cxcl2, and reduced cytokine-induced ERK phosphorylation. Further, NF-κB genomic iNos promoter binding was reduced by 50%, and NF-κB-dependent mRNA expression was blocked. These effects were associated with NF-κB subunit p65 hyperacetylation. Taken together, these data provide a rationale for clinical trials of safety and efficacy of KDACi in patients with autoimmune disease such as type 1 diabetes.
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http://dx.doi.org/10.1073/pnas.1320850111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903225PMC
January 2014

Histone deacetylase inhibitors for treating a spectrum of diseases not related to cancer.

Mol Med 2011 May-Jun;17(5-6):333-52. Epub 2011 May 5.

Department of Medicine, Division of Infectious Diseases, University of Colorado Denver, Aurora, Colorado 80045, USA.

This issue of Molecular Medicine contains 14 original research reports and state-of-the-art reviews on histone deacetylase inhibitors (HDACi's), which are being studied in models of a broad range of diseases not related to the proapoptotic properties used to treat cancer. The spectrum of these diseases responsive to HDACi's is for the most part due to several antiinflammatory properties, often observed in vitro but importantly also in animal models. One unifying property is a reduction in cytokine production as well as inhibition of cytokine postreceptor signaling. Distinct from their use in cancer, the reduction in inflammation by HDACi's is consistently observed at low concentrations compared with the higher concentrations required for killing tumor cells. This characteristic makes HDACi's attractive candidates for treating chronic diseases, since low doses are well tolerated. For example, low oral doses of the HDACi givinostat have been used in children to reduce arthritis and are well tolerated. In addition to the antiinflammatory properties, HDACi's have shown promise in models of neurodegenerative disorders, and HDACi's also hold promise to drive HIV-1 out of latently infected cells. No one molecular mechanism accounts for the non-cancer-related properties of HDACi's, since there are 18 genes coding for histone deacetylases. Rather, there are mechanisms unique for the pathological process of specific cell types. In this overview, we summarize the preclinical data on HDACi's for therapy in a wide spectrum of diseases unrelated to the treatment of cancer. The data suggest the use of HDACi's in treating autoimmune as well as chronic inflammatory diseases.
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http://dx.doi.org/10.2119/molmed.2011.00116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105126PMC
October 2011

Pharmacokinetics, safety and inducible cytokine responses during a phase 1 trial of the oral histone deacetylase inhibitor ITF2357 (givinostat).

Mol Med 2011 May-Jun;17(5-6):353-62. Epub 2011 Feb 22.

Department of Medicine, University of Colorado Denver, Aurora, Colorado 80045, USA.

ITF2357 (givinostat) is a histone deacetylase inhibitor with antiinflammatory properties at low nanomolar concentrations. We report here a phase I safety and pharmacokinetics trial in healthy males administered 50, 100, 200, 400 or 600 mg orally. After 50 mg, mean maximal plasma concentrations reached 104 nmol/L 2 h after dosing, with a half-life of 6.9 h. After 100 mg, maximal concentration reached 199 nmol/L at 2.1 h with a half-life of 6.0 h. Repeat doses for 7 consecutive days of 50, 100 or 200 mg resulted in nearly the same kinetics. There were no serious adverse effects (AEs) and no organ toxicities. However, there was a dose-dependent but transient fall in platelets. After 7 daily doses of 50 or 100 mg, the mean decrease in platelets of 17 and 25% was not statistically significant and returned to baseline within 14 d. Blood removed from the subjects after oral dosing was cultured ex vivo with endotoxin, and the release of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-1Ra, interferon (IFN)-γ and IL-10 was determined. Maximal reduction in IL-1β, TNFα, IL-6 and IFNγ was observed 4 h after dosing but returned to baseline at 12 h. There was no significant reduction in IL-1Ra or IL-10. With daily dosing, the fall in cytokine production in blood cultures observed on day 7 was nearly the same as that of the first day. We conclude that dosing of 50 or 100 mg ITF2357 is safe in healthy humans and transiently but repeatedly reduces the production of proinflammatory cytokines without affecting production of antiinflammatory cytokines.
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http://dx.doi.org/10.2119/molmed.2011.00020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105139PMC
October 2011

The histone deacetylase inhibitor ITF2357 decreases surface CXCR4 and CCR5 expression on CD4(+) T-cells and monocytes and is superior to valproic acid for latent HIV-1 expression in vitro.

J Acquir Immune Defic Syndr 2010 May;54(1):1-9

Department of Medicine, University of Colorado Denver, 12700 E. 19th Avenue B-168, Aurora, CO 80045, USA.

Objectives: Chromatin-associated repression is one mechanism that maintains HIV-1 latency. Inhibition of histone deacetylases (HDAC) reverses this repression resulting in viral expression from quiescently infected cells. Clinical studies with the HDAC inhibitor valproic acid (VPA) failed to substantially decrease the latent pool within resting CD4(+) cells. Here we compared the efficacy of ITF2357, an orally active and safe HDAC inhibitor, with VPA for HIV-1 expression from latently infected cells in vitro. We also evaluated the effect of ITF2357 on the surface expression of CXCR4 and CCR5.

Methods: Latently infected cell lines were incubated with either ITF2357 or VPA and p24 levels were measured. Peripheral blood mononuclear cells of uninfected donors were treated with ITF2357 and HIV-1 coreceptors expression was assessed by flow cytometry.

Results: At clinically relevant concentrations, ITF2357 increased p24 by 15-fold in ACH2 cells and by 9-fold in U1 cells, whereas VPA increased expression less than 2-fold. Analogues of ITF2357 primarily targeting HDAC-1 increased p24 up to 30-fold. In CD4(+) T cells treated with ITF2357, CXCR4 expression decreased by 54% (P < 0.001).

Conclusion: ITF2357 is superior to VPA in inducing HIV-1 from latently infected cells. Safely used in humans, ITF2357 is an attractive candidate for HIV-1 clinical purging.
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http://dx.doi.org/10.1097/QAI.0b013e3181d3dca3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3534976PMC
May 2010

Pleiotropic anti-myeloma activity of ITF2357: inhibition of interleukin-6 receptor signaling and repression of miR-19a and miR-19b.

Haematologica 2010 Feb 27;95(2):260-9. Epub 2009 Aug 27.

Department of Medical Sciences, University of Milan, Fondazione IRCCS Policlinico, Milano, Italy.

Background: The histone deacetylase inhibitor ITF2357 has potent cytotoxic activity in multiple myeloma in vitro and has entered clinical trials for this disease.

Design And Methods: In order to gain an overall view of the activity of ITF2357 and identify specific pathways that may be modulated by the drug, we performed gene expression profiling of the KMS18 multiple myeloma cell line treated with the drug. The modulation of several genes and their biological consequence were verified in a panel of multiple myeloma cell lines and cells freshly isolated from patients by using polymerase chain reaction analysis and western blotting.

Results: Out of 38,500 human genes, we identified 140 and 574 up-regulated genes and 102 and 556 down-modulated genes at 2 and 6 h, respectively, with a significant presence of genes related to transcription regulation at 2 h and to cell cycling and apoptosis at 6 h. Several of the identified genes are particularly relevant to the biology of multiple myeloma and it was confirmed that ITF2357 also modulated their encoded proteins in different multiple myeloma cell lines. In particular, ITF2357 down-modulated the interleukin-6 receptor alpha (CD126) transcript and protein in both cell lines and freshly isolated patients' cells, whereas it did not significantly modify interleukin-6 receptor beta (CD130) expression. The decrease in CD126 expression was accompanied by decreased signaling by interleukin-6 receptor, as measured by STAT3 phosphorylation in the presence and absence of inter-leukin-6. Finally, the drug significantly down-modulated the MIRHG1 transcript and its associated microRNA, miR-19a and miR-19b, known to have oncogenic activity in multiple myeloma.

Conclusions: ITF2357 inhibits several signaling pathways involved in myeloma cell growth and survival.
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http://dx.doi.org/10.3324/haematol.2009.012088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817029PMC
February 2010

Histone deacetylase (HDAC) inhibitors reduce the glial inflammatory response in vitro and in vivo.

Neurobiol Dis 2009 Nov 25;36(2):269-79. Epub 2009 Jul 25.

Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy.

Histone deacetylase inhibitors (HDACi) are emerging tools for epigenetic modulation of gene expression and suppress the inflammatory response in models of systemic immune activation. Yet, their effects within the brain are still controversial. Also, whether HDACs are expressed in astrocytes or microglia is unclear. Here, we report the identification of transcripts for HDAC 1-11 in cultured mouse glial cells. Two HDACi such as SAHA and ITF2357 induce dramatic increase of histone acetylation without causing cytotoxicity of cultured cells. Of note, the two compounds inhibit expression of pro-inflammatory mediators by LPS-challenged glial cultures, and potentiate immunosuppression triggered by dexamethasone in vitro. The anti-inflammatory effect is not due to HDACi-induced transcription of immunosuppressant proteins, (including SOCS-1/3) or microRNA-146. Rather, it is accompanied by direct alteration of transcription factor DNA binding and ensuing transcriptional activation. Indeed, both HDACi impair NFkappaB-dependent IkappaBalpha resynthesis in glial cells exposed to LPS, and, among various AP1 subunits and NFkappaB p65, affect the DNA binding activity of c-FOS, c-JUN and FRA2. Importantly, ITF2357 reduces the expression of pro-inflammatory mediators in the striatum of mice iontophoretically injected with LPS. Data demonstrate that mouse glial cells have ongoing HDAC activity, and its inhibition suppresses the neuroinflammatory response because of a direct impairment of the transcriptional machinery.
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http://dx.doi.org/10.1016/j.nbd.2009.07.019DOI Listing
November 2009

A PEGylated Fab' fragment against tumor necrosis factor for the treatment of Crohn disease: exploring a new mechanism of action.

BioDrugs 2008 ;22(5):331-7

Inflammation Discovery, UCB Celltech, Slough, UK.

Antibodies, having a high specificity for their particular target, are increasingly being used as therapeutic agents with functions including agonist, antagonist, and targeted drug delivery. The use of many biologic therapies, including antibody fragments, is generally limited by their rapid clearance from plasma. A commonly used approach to extend exposure to biologic therapies is the attachment of polyethylene glycol.Tumor necrosis factor (TNF)-alpha is a multifunctional cytokine involved in the regulation of immune responses. Elevated levels of TNFalpha are found in a wide range of diseases, including the chronic inflammatory conditions rheumatoid arthritis, psoriasis, and Crohn disease (CD). Anti-TNFalpha antibodies have proved highly efficacious in the treatment of these conditions. In addition, they have proved invaluable for investigating the role of TNFalpha in disease etiology. Based on evidence showing that neutralizing antibodies to TNFalpha were effective in animal models of CD, anti-TNFalpha antibody treatments were assessed in clinical trials. Interestingly, the anti-TNFalpha antibody etanercept proved ineffective at achieving remission in active CD despite potently neutralizing soluble TNFalpha. This indicated that an additional mode of action is also involved in the efficacy of the anti-TNFalpha agents adalimumab, certolizumab pegol, and infliximab in CD; one suggestion was apoptosis. However, etanercept, like adalimumab and infliximab, can induce apoptosis. Furthermore, certolizumab pegol (which has demonstrated efficacy in CD) does not cause complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, apoptosis, or necrosis of neutrophils, all measured in vitro. These functional differences observed with certolizumab pegol stem from its unique structure that does not include the crystallizable fragment (Fc) portion present in the other anti-TNFalpha agents, and the way in which it signals through membrane TNF. It is well established that bacteria are a major part of the inflammatory process in CD. The property identified that reflected the efficacies of the anti-TNFalpha agents etanercept, adalimumab, certolizumab pegol, and infliximab in CD was the ability to inhibit the cytokine production by monocytes that is induced by bacterial lipopolysaccharide. It may therefore be the case that this mode of action is important for efficacy in CD.
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http://dx.doi.org/10.2165/00063030-200822050-00005DOI Listing
November 2008

ATP is released by monocytes stimulated with pathogen-sensing receptor ligands and induces IL-1beta and IL-18 secretion in an autocrine way.

Proc Natl Acad Sci U S A 2008 Jun 3;105(23):8067-72. Epub 2008 Jun 3.

Cell Biology Unit, National Cancer Research Institute, Largo Rosanna Benzi 10, 16132 Genoa, Italy.

IL-1beta and IL-18 are crucial mediators of inflammation, and a defective control of their release may cause serious diseases. Yet, the mechanisms regulating IL-1beta and IL-18 secretion are partially undefined. Both cytokines are produced as inactive cytoplasmic precursors. Processing to the active form is mediated by caspase-1, which is in turn activated by the multiprotein complex inflammasome. Here, we show that in primary human monocytes microbial components acting on different pathogen-sensing receptors and the danger-associated molecule uric acid are all competent to induce maturation and secretion of IL-1beta and IL-18 through a process that involves as a first event the extracellular release of endogenous ATP. ATP release is followed by autocrine stimulation of the purinergic receptors P2X(7). Indeed, antagonists of the P2X(7) receptor (P2X(7)R), or treatment with apyrase, prevent IL-1beta and IL-18 maturation and secretion triggered by the different stimuli. At variance, blocking P2X(7)R activity has no effects on IL-1beta secretion by monocytes carrying a mutated inflammasome that does not require exogenous ATP for activation. P2X(7)R engagement is followed by K+ efflux and activation of phospholipase A(2). Both events are required for processing and secretion induced by all of the stimuli. Thus, stimuli acting on different pathogen-sensing receptors converge on a common pathway where ATP externalization is the first step in the cascade of events leading to inflammasome activation and IL-1beta and IL-18 secretion.
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http://dx.doi.org/10.1073/pnas.0709684105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430360PMC
June 2008

ITF1697, a stable Lys-Pro-containing peptide, inhibits weibel-palade body exocytosis induced by ischemia/reperfusion and pressure elevation.

Mol Med 2007 Nov-Dec;13(11-12):615-24

CNR Institute of Clinical Physiology, Medical School, University of Pisa, Pisa, Italy.

A number of Lys-Pro-containing short peptides have been described as possessing a variety of biological activities in vitro. Because of limited metabolic stability, however, their efficacy in vivo is uncertain. To exploit the pharmacological potential of Lys-Pro-containing short peptides, we synthesized a series of chemically modified forms of these peptides. One of them, ITF1697 (Gly-(Nalpha-Et)Lys-Pro-Arg) was stable in vivo and particularly efficacious in experimental models of disseminated endotoxemia and of cardiovascular disorders. Using intravital fluorescence microscopy, we studied the peptide cellular and molecular basis of protection in the Syrian hamster cheek pouch microcirculation subjected to ischemia/reperfusion (I/R) and in pressure elevation-induced proinflammatory responses in isolated Sprague-Dawley rat lungs. Continuous intravenous infusion of ITF1697 at 0.1 to 100 mug/kg/min nearly completely protected the cheek pouch microcirculation from I/R injury as measured by decreased vascular permeability and increased capillary perfusion. Adhesion of leukocytes and platelets to blood vessels was strongly inhibited by the peptide. ITF1697 exerted its activity at the early stages of endothelial activation and inhibited P-selectin and von Willebrand factor secretion. Further mechanistic studies in the rat lung preparation revealed that the peptide inhibited the intracellular Ca(2+)-dependent fusion of Weibel-Palade bodies with the plasma membrane. The ability of ITF1697 to inhibit the early functions of activated endothelial cells, such as the exocytosis of Weibel-Palade bodies, represents a novel and promising pharmacological tool in model of pathologies of a variety of microvascular disorders.
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http://dx.doi.org/10.2119/2007-00079.BertugliaDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2034195PMC
February 2008

Mechanism of action of certolizumab pegol (CDP870): in vitro comparison with other anti-tumor necrosis factor alpha agents.

Inflamm Bowel Dis 2007 Nov;13(11):1323-32

Inflammation Discovery, UCB Celltech, Slough, UK.

Background: Inhibitors of tumor necrosis factor alpha (TNFalpha) have demonstrated significant efficacy in chronic inflammatory diseases, including Crohn's disease (CD). To further elucidate the mechanisms of action of these agents, we compared the anti-TNFalpha agents certolizumab pegol, infliximab, adalimumab, and etanercept in several in vitro systems.

Methods: The ability of each anti-TNFalpha agent to neutralize soluble and membrane-bound TNFalpha; mediate cytotoxicity, affect apoptosis of activated human peripheral blood lymphocytes and monocytes; induce degranulation of human peripheral blood granulocytes, and modulate lipopolysaccharide (LPS)-induced interleukin (IL)-1beta production by human monocytes was measured in vitro.

Results: All 4 agents neutralized soluble TNFalpha and bound to and neutralized membrane TNFalpha. Infliximab and adalimumab were comparable in their ability to mediate complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and to increase the proportion of cells undergoing apoptosis and the level of granulocyte degranulation. Etanercept generally mediated these effects to a lesser degree, while certolizumab pegol gave similar results to the control reagents. LPS-induced IL-1beta production was inhibited by certolizumab pegol, infliximab, and adalimumab, but only partially inhibited by etanercept.

Conclusions: In contrast to the other anti-TNFalpha agents tested, certolizumab pegol did not mediate increased levels of apoptosis in any of the in vitro assays used, suggesting that these mechanisms are not essential for the efficacy of anti-TNFalpha agents in CD. As certolizumab pegol, infliximab, and adalimumab, but not etanercept, almost completely inhibited LPS-induced IL-1beta release from monocytes, inhibition of cytokine production may be important for efficacy of anti-TNFalpha agents in CD.
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http://dx.doi.org/10.1002/ibd.20225DOI Listing
November 2007

Pharmacological inhibition of histone deacetylases by suberoylanilide hydroxamic acid specifically alters gene expression and reduces ischemic injury in the mouse brain.

Mol Pharmacol 2006 Dec 31;70(6):1876-84. Epub 2006 Aug 31.

Department of Preclinical and Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Firenze, Italy.

Pharmacological manipulation of gene expression is considered a promising avenue to reduce postischemic brain damage. Histone deacetylases (HDACs) play a central role in epigenetic regulation of transcription, and inhibitors of HDACs are emerging as neuroprotective agents. In this study, we investigated the effect of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on histone acetylation in control and ischemic mouse brain. We report that brain histone H3 acetylation was constitutively present at specific lysine residues in neurons and astrocytes. It is noteworthy that in the ischemic brain tissue subjected to 6 h of middle cerebral artery occlusion, histone H3 acetylation levels drastically decreased, without evidence for a concomitant change of histone acetyl-transferase or deacetylase activities. Treatment with SAHA (50 mg/kg i.p.) increased histone H3 acetylation within the normal brain (of approximately 8-fold after 6 h) and prevented histone deacetylation in the ischemic brain. These effects were accompanied by increased expression of the neuroprotective proteins Hsp70 and Bcl-2 in both control and ischemic brain tissue 24 h after the insult. It is noteworthy that at the same time point, mice injected with SAHA at 25 and 50 mg/kg had smaller infarct volumes compared with vehicle-receiving animals (28.5% and 29.8% reduction, p < 0.05 versus vehicle, Student's t test). At higher doses, SAHA was less efficient in increasing Bcl-2 and Hsp70 expression and did not afford significant ischemic neuroprotection (13.9% infarct reduction). Data demonstrate that pharmacological inhibition of HDACs promotes expression of neuroprotective proteins within the ischemic brain and underscores the therapeutic potential of molecules inhibiting HDACs for stroke therapy.
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http://dx.doi.org/10.1124/mol.106.027912DOI Listing
December 2006

Histone deacetylase inhibitors prevent exocytosis of interleukin-1beta-containing secretory lysosomes: role of microtubules.

Blood 2006 Sep 9;108(5):1618-26. Epub 2006 May 9.

Laboratory of Experimental Oncology E, National Cancer Research Institute, Genoa, Italy.

A number of agents reducing interleukin-1beta (IL-1beta) activity are being developed as novel immunomodulatory and anti-inflammatory therapies. However, the elucidation of their molecular mechanism of action is required in the context of medical management of inflammatory diseases. Inhibitors of histone deacetylases (HDACs) are promising anticancer agents with pleiotropic activities. Of these, suberoylanilide hydroxamic acid has been reported to inhibit the production of several proinflammatory cytokines. In the present study, we investigated the effects of 2 HDAC inhibitors on IL-1beta secretion: suberoylanilide hydroxamic acid and a newly developed hydroxamic acid-derived compound ITF2357. These HDAC inhibitors do not affect the synthesis or intracellular localization of IL-1beta but both strongly reduce the levels of extracellular IL-1beta by preventing the exocytosis of IL-1beta-containing secretory lysosomes. At nanomolar concentrations, ITF2357 reduces the secretion of IL-1beta following ATP activation of the P2X7 receptor. Whereas the inhibition of HDACs results in hyperacetylation of tubulin, acetylation of HSP90 was unaffected. The reduction in IL-1beta secretion appears to be due to disruption of microtubules impairing lysosome exocytosis. Together, these observations indicate that a functional microtubule network is required for IL-1beta secretion and suggest that disruption of tubulin is the mechanism by which inhibitors of HDACs reduce the secretion of IL-1beta.
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http://dx.doi.org/10.1182/blood-2006-03-014126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1895509PMC
September 2006

The histone deacetylase inhibitor ITF2357 reduces production of pro-inflammatory cytokines in vitro and systemic inflammation in vivo.

Mol Med 2005 Jan-Dec;11(1-12):1-15

Research Center, Italfarmaco, Cinisello Balsamo, Milan, Italy.

We studied inhibition of histone deacetylases (HDACs), which results in the unraveling of chromatin, facilitating increased gene expression. ITF2357, an orally active, synthetic inhibitor of HDACs, was evaluated as an anti-inflammatory agent. In lipopolysaccharide (LPS)-stimulated cultured human peripheral blood mononuclear cells (PBMCs), ITF2357 reduced by 50% the release of tumor necrosis factor-alpha (TNFalpha) at 10 to 22 nM, the release of intracellular interleukin (IL)-1alpha at 12 nM, the secretion of IL-1beta at 12.5 to 25 nM, and the production of interferon-gamma (IFNgamma) at 25 nM. There was no reduction in IL-8 in these same cultures. Using the combination of IL-12 plus IL-18, IFNgamma and IL-6 production was reduced by 50% at 12.5 to 25 nM, independent of decreased IL-1 or TNFalpha. There was no evidence of cell death in LPS-stimulated PBMCs at 100 nM ITF2357, using assays for DNA degradation, annexin V, and caspase-3/7. By Northern blotting of PBMCs, there was a 50% to 90% reduction in LPS-induced steady-state levels of TNFalpha and IFNgamma mRNA but no effect on IL-1beta or IL-8 levels. Real-time PCR confirmed the reduction in TNFalpha RNA by ITF2357. Oral administration of 1.0 to 10 mg/kg ITF2357 to mice reduced LPS-induced serum TNFalpha and IFNgamma by more than 50%. Anti-CD3-induced cytokines were not suppressed by ITF2357 in PBMCs either in vitro or in the circulation in mice. In concanavalin-A-induced hepatitis, 1 or 5 mg/kg of oral ITF2357 significantly reduced liver damage. Thus, low, nonapoptotic concentrations of the HDAC inhibitor ITF2357 reduce pro-inflammatory cytokine production in primary cells in vitro and exhibit anti-inflammatory effects in vivo.
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http://dx.doi.org/10.2119/2006-00005.DinarelloDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449516PMC
June 2009

The Mycobacterium tuberculosis chaperonin 10 monomer exhibits structural plasticity.

Biopolymers 2004 Oct;75(2):148-62

Italfarmaco Research Centre, via Lavoratori 54, Cinisello Balsamo 20092 Milan, Italy.

The conditions which favor dissociation of oligomeric Mycobacterium tuberculosis chaperonin 10 and the solution structure of the monomer were studied by analytical ultracentrifugation, size exclusion chromatography, fluorescence, and circular dichroism spectroscopies. At neutral pH and in the absence of divalent cations, the protein is fully monomeric below approximately a 4.7 microM concentration. Under these conditions the monomer forms completely unfolded and partially folded conformers which are in equilibrium with each other. One conformer accumulates over the others which is stable within a very narrow range of temperatures. It contains a beta-sheet-structured C-terminal half and a mostly disordered N-terminal half. Other components of the equilibrium include partially helical structures which do not completely unfold at high temperature or under strong acidic conditions. Complete unfolding of the monomer occurs in the presence of denaturants or below 14 degrees C. Cold-denaturation is detected at an unusually high temperature and this may be due to the concentration of hydrophobic residues, which is larger in chaperonins than in other globular proteins. Finally, the monomer self-associates in the pH range 5.8-2.9, where it forms small oligomers. A structure-activity relationship was investigated with the sequences known to be involved in the various biological activities of the monomer.
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http://dx.doi.org/10.1002/bip.20106DOI Listing
October 2004

Mycobacterium tuberculosis chaperonin 10 is secreted in the macrophage phagosome: is secretion due to dissociation and adoption of a partially helical structure at the membrane?

J Bacteriol 2003 Jul;185(14):4256-67

Italfarmaco Research Centre, Cinisello Balsamo 20092, Milan, Italy.

To confirm that Mycobacterium tuberculosis chaperonin 10 (Cpn10) is secreted outside the live bacillus, infected macrophages were examined by electron microscopy. This revealed that the mycobacterial protein accumulates both in the wall of the bacterium and in the matrix of the phagosomes in which ingested mycobacteria survive within infected macrophages. To understand the structural implications underlying this secretion, a structural study of M. tuberculosis Cpn10 was performed under conditions that are generally believed to mimic the membrane environment. It was found that in buffer-organic solvent mixtures, the mycobacterial protein forms two main species, namely, a partially helical monomer that prevails in dilute solutions at room temperature and a dimer that folds into a beta-sheet-dominated structure and prevails in either concentrated protein solutions at room temperature or in dilute solutions at low temperature. A partially helical monomer was also found and was completely associated with negatively charged detergents in a micelle-bound state. Remarkably, zwitterionic lipids had no effect on the protein structure. By using N- and C-truncated forms of the protein, the C- and N-terminal sequences were identified as possessing an amphiphilic helical character and as selectively associating with acidic detergent micelles. When the study was extended to other chaperonins, it was found that human Cpn10 is also monomeric and partially helical in dilute organic solvent-buffer mixtures. In contrast, Escherichia coli Cpn10 is mostly dimeric and predominately beta-sheet in both dilute and concentrated solutions. Interestingly, human Cpn10 also crosses biological membranes, whereas the E. coli homologue is strictly cytosolic. These results suggest that dissociation to partially helical monomers and interaction with acidic lipids may be two important steps in the mechanism of secretion of M. tuberculosis Cpn10 to the external environment.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC164881PMC
http://dx.doi.org/10.1128/JB.185.14.4256-4267.2003DOI Listing
July 2003

Mycobacterium tuberculosis chaperonin 10 heptamers self-associate through their biologically active loops.

J Bacteriol 2003 Jul;185(14):4172-85

Medical Microbiology, Department of Cellular and Molecular Medicine, St. George's Hospital Medical School, London SW17 0RE, England.

The crystal structure of Mycobacterium tuberculosis chaperonin 10 (cpn10(Mt)) has been determined to a resolution of 2.8 A. Two dome-shaped cpn10(Mt) heptamers complex through loops at their bases to form a tetradecamer with 72 symmetry and a spherical cage-like structure. The hollow interior enclosed by the tetradecamer is lined with hydrophilic residues and has dimensions of 30 A perpendicular to and 60 A along the sevenfold axis. Tetradecameric cpn10(Mt) has also been observed in solution by dynamic light scattering. Through its base loop sequence cpn10(Mt) is known to be the agent in the bacterium responsible for bone resorption and for the contribution towards its strong T-cell immunogenicity. Superimposition of the cpn10(Mt) sequences 26 to 32 and 66 to 72 and E. coli GroES 25 to 31 associated with bone resorption activity shows them to have similar conformations and structural features, suggesting that there may be a common receptor for the bone resorption sequences. The base loops of cpn10s in general also attach to the corresponding chaperonin 60 (cpn60) to enclose unfolded protein and to facilitate its correct folding in vivo. Electron density corresponding to a partially disordered protein subunit appears encapsulated within the interior dome cavity of each heptamer. This suggests that the binding of substrates to cpn10 is possible in the absence of cpn60.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC164875PMC
http://dx.doi.org/10.1128/JB.185.14.4172-4185.2003DOI Listing
July 2003

The mitochondrial network of human neutrophils: role in chemotaxis, phagocytosis, respiratory burst activation, and commitment to apoptosis.

J Immunol 2003 Feb;170(4):1964-72

Department of Medicine, School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, UK.

It is commonly assumed that human neutrophils possess few, if any, functional mitochondria and that they do not depend on these organelles for cell function. We have used the fluorescent mitochondrial indicators, JC-1, MitoTracker Red, and dihydrorhodamine 123 to show that live neutrophils possess a complex mitochondrial network that extends through the cytoplasm. The membrane potential of these mitochondria was rapidly (within 2 min) disrupted by the addition of FCCP (IC(50) = 20 nM), but not by the Fo-ATPase inhibitor, oligomycin (at up to 7 microg/ml). However, inhibition of mitochondrial function with both agents resulted in cell shape changes. Neither activation of the respiratory burst nor phagocytosis of either latex particles or serum-opsonized Staphylococcus aureus was affected by the addition of FCCP or oligomycin. However, FCCP inhibited chemotaxis at concentrations that paralleled disruption of mitochondrial membrane potential. Furthermore, prolonged (2-h) incubation with oligomycin resulted in an impaired ability to activate a respiratory burst and also inhibited chemotaxis. These observations indicate that intact mitochondrial function is required to sustain some neutrophil functions, but not for the rapid initiation of the respiratory burst or phagocytosis. Loss of mitochondrial membrane potential was a very early marker for commitment of neutrophils into apoptosis and preceded the appearance of phosphatidylserine on the cell surface. However, inhibition of mitochondrial function did not accelerate the rate of neutrophil apoptosis. These data shed important insights into the hitherto unrecognized importance of mitochondria in the function of neutrophils during infection and inflammation.
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http://dx.doi.org/10.4049/jimmunol.170.4.1964DOI Listing
February 2003

Differential role of neutrophil Fcgamma receptor IIIB (CD16) in phagocytosis, bacterial killing, and responses to immune complexes.

Arthritis Rheum 2002 May;46(5):1351-61

University of Liverpool, Liverpool, UK.

Objective: To determine the roles played by the neutrophil Fcgamma receptor type II (FcgammaRII) (CD32) and FcgammaRIIIb (CD16) in phagocytosis, bacterial killing, and activation by immune complexes (ICs) and to test the hypothesis that inhibition of pathologic effector neutrophil function is possible without compromising host defense.

Methods: Receptor function was probed by enzymic removal of FcgammaRIIIb from the cell surface and by use of Fab/F(ab')(2) fragments of monoclonal antibodies to block receptor-ligand binding. Cells were challenged with (a) serum-opsonized Staphylococcus aureus, (b) serum- and IgG-opsonized latex particles, and (c) synthetic soluble and insoluble ICs to mimic bacterial and inflammatory stimuli.

Results: Phosphatidylinositol-phospholipase C treatment removed >97% of surface FcgammaRIIIb from neutrophils previously treated with tumor necrosis factor alpha to mobilize intracellular stores of receptor. This treatment profoundly inhibited activation of primed neutrophils by soluble ICs of the type found in diseased rheumatoid joints, but had no effect on phagocytosis and killing of serum-opsonized S aureus.

Conclusion: FcgammaRIIIb plays a major role in the secretion of toxic products in response to ICs, but little or no role in the phagocytosis and killing of serum-opsonized bacteria. The selective suppression of effector neutrophil function is therefore possible. FcgammaRIIIb, or its intracellular signaling pathway, is a potential therapeutic target in inflammatory diseases such as rheumatoid arthritis, because disruption of its function should decrease inflammatory tissue damage, but not jeopardize host protection against infection.
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http://dx.doi.org/10.1002/art.10230DOI Listing
May 2002