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Histone acetyltransferases (HATs) are important mediators of epigenetic post-translational modifications of histones that play important roles in health and disease. A disturbance of these modifications can result in disease states, such as cancer or inflammatory diseases. Inhibitors of HATs (HATi) such as lysine (K) acetyltransferase 8 (KAT8), could be used to study the epigenetic processes in diseases related to these enzymes or to investigate HATs as therapeutic targets. However, the development of HATi is challenged by the difficulties in kinetic characterization of HAT enzymes and their inhibitors to enable calculation of a reproducible inhibitory potency. In this study, a fragment screening approach was used, enabling identification of 4-amino-1-naphthol, which potently inhibited KAT8. The inhibitor was investigated for enzyme inhibition using kinetic and calorimetric binding studies. This allowed for calculation of the K(i) values for both the free enzyme as well as the acetylated intermediate. Importantly, it revealed a striking difference in binding affinity between the acetylated enzyme and the free enzyme, which could not be revealed by the IC(50) value. This shows that kinetic characterization of inhibitors and calculation of K(i) values is crucial for determining the binding constants of HAT inhibitors. We anticipate that more comprehensive characterization of enzyme inhibition, as described here, is needed to advance the field of HAT inhibitors.

Transcriptional deregulation emerges as a key pathogenetic mechanism in ALS pathogenesis, and non-class-specific histone deacetylase (HDACs) inhibitors proved of therapeutic efficacy in preclinical models of ALS. When tested in patients, however, these drugs failed, probably because of a lack of selectivity toward pathogenetic HDACs. Here, we studied the effects of MC1568, an inhibitor of Class-II HDACs which have been reported to contribute to ALS pathogenesis. We focused on transcriptional regulation of glutamate transporter EAAT2, whose reduced expression may contribute to motor neuron degeneration in ALS. We report that MC1568 highly increased EAAT2 transcripts in primary cultures of mouse glia, but these increases did not correlate with increased glutamate uptake capacity. Accordingly, we found that MC1568 augmented protein expression of EAAT2 together with its sumoylation, a post-translational modification typically altering protein function and localization. When tested in SOD1G93A mice, however, MC1568 fully restored the reduced spinal cord expression of EAAT2 and glutamate uptake up to control levels. A prolonged treatment with MC1568 (from onset to end stage) was unable to prolong survival of mice. Data reveal a key role of Class-II HDACs in expression and function of glutamate transporter, further corroborating preclinical and clinical evidence that the sole restoration of glutamate uptake is not of therapeutic relevance to ALS therapy.

Sirt2 is a target for the treatment of neurological, metabolic, and age-related diseases including cancer. Here we report a series of Sirt2 inhibitors based on the 1,2,4-oxadiazole scaffold. These compounds are potent Sirt2 inhibitors active at single-digit μM level by using the Sirt2 substrate α-tubulin-acetylLys40 peptide and inactive up to 100 μM against Sirt1, -3, and -5 (deacetylase and desuccinylase activities). Their mechanism of inhibition is uncompetitive toward both the peptide substrate and NAD(+), and the crystal structure of a 1,2,4-oxadiazole analog in complex with Sirt2 and ADP-ribose reveals its orientation in a still unexplored subcavity useful for further inhibitor development. Tested in leukemia cell lines, 35 and 39 induced apoptosis and/or showed antiproliferative effects at 10 or 25 μM after 48 h. Western blot analyses confirmed the involvement of Sirt2 inhibition for their effects in NB4 and in U937 cells. Our results provide novel Sirt2 inhibitors with a compact scaffold and structural insights for further inhibitor improvement.

Human p300 is a polyhedric transcriptional coactivator that plays a crucial role in acetylating histones on specific lysine residues. A great deal of evidence shows that p300 is involved in several diseases, including leukemia, tumors, and viral infection. Its involvement in pleiotropic biological roles and connections to diseases provide the rationale to determine how its modulation could represent an amenable drug target. Several p300 inhibitors (i.e., histone acetyltransferase inhibitors, HATis) have been described so far, but they all suffer from low potency, lack of specificity, or low cell permeability, which thus highlights the need to find more effective inhibitors. Our cinnamoyl derivative, 2,6-bis(3-bromo-4-hydroxybenzylidene) cyclohexanone (RC56), was identified as an active and selective p300 inhibitor and was proven to be a good hit candidate to investigate the structure-activity relationship toward p300. Herein, we describe the design, synthesis, and biological evaluation of new HATis structurally related to our hit; moreover, we investigate the interactions between p300 and the best-emerged hits by means of induced-fit docking and molecular-dynamics simulations, which provided insight into the peculiar chemical features that influence their activity toward the targeted enzyme.

Introduction: The histone methyltransferase EZH2 is the catalytic subunit of the PRC2 complex involved in H3K27 trimethylation. Aberrant PRC2 activity has been reported in several cancers and EZH2 overexpression has been associated with poor outcome in different tumors. EZH2 somatic mutations and deletions was found in lymphomas, myelodysplastic and myeloproliferative disorders and associated with higher H3K27me3 levels. Numerous chemical entities have been studied as EZH2 inhibitors in the recent years and some of them entered the cancer clinical arena. Areas covered: This review summarizes recent efforts in the drug development of EZH2 inhibitors reported in the patent literature covering the 2014–2016 period, and their potential use as therapeutics mainly in cancerous diseases. Expert opinion: Despite the number of compounds described, only a few of them entered the clinical arena. Moreover, most of the compounds developed share a common 2-pyridone ring pharmacophore. Recently, secondary mutants have been described to be resistant to the standard EZH2 inhibitors treatment. Based on these data a lot of effort is still required to find new chemical entities that inhibit EZH2 directly, or indirectly (via PRC2 disruption). Several issues are still to be settled, such as drug resistance and the importance of selectivity over EZH1 or somatic EZH2 mutants.

A comprehensive study on essential oils extracted from different Calamintha nepeta (L.) Savi subsp. glandulosa (Req.) Ball samples from Tarquinia (Italy) is reported. In this study, the 24-h steam distillation procedure for essential oil preparation, in terms of different harvesting and extraction times, was applied. The Gas chromatography–mass spectrometry (GC/MS) analysis showed that C. nepeta (L.) Savi subsp. glandulosa (Req.) Ball essential oils from Tarquinia belong to the pulegone-rich chemotype. The analysis of 44 samples revealed that along with pulegone, some other chemicals may participate in exerting the related antifungal activity. The results indicated that for higher activity, the essential oils should be produced with at least a 6-h steam distillation process. Even though it is not so dependent on the period of harvesting, it could be recommended not to harvest the plant in the fruiting stage, since no significant antifungal effect was shown. The maximum essential oil yield was obtained in August, with the highest pulegone percentage. To obtain the oil with a higher content of menthone, September and October should be considered as the optimal periods. Regarding the extraction duration, vegetative stage material gives the oil in the first 3 h, while material from the reproductive phase should be extracted at least at 6 or even 12 h.

SHH Medulloblastoma (SHH-MB) is a pediatric brain tumor characterized by an inappropriate activation of the developmental Hedgehog (Hh) signaling. SHH-MB patients treated with the FDA-approved vismodegib, an Hh inhibitor that targets the transmembrane activator Smoothened (Smo), have shown the rapid development of drug resistance and tumor relapse due to novel Smo mutations. Moreover, a subset of patients did not respond to vismodegib because mutations were localized downstream of Smo. Thus, targeting downstream Hh components is now considered a preferable approach. We show here that selective inhibition of the downstream Hh effectors HDAC1 and HDAC2 robustly counteracts SHH-MB growth in mouse models. These two deacetylases are upregulated in tumor and their knockdown inhibits Hh signaling and decreases tumor growth. We demonstrate that mocetinostat (MGCD0103), a selective HDAC1/HDAC2 inhibitor, is a potent Hh inhibitor and that its effect is linked to Gli1 acetylation at K518. Of note, we demonstrate that administration of mocetinostat to mouse models of SHH-MB drastically reduces tumor growth, by reducing proliferation and increasing apoptosis of tumor cells and prolongs mouse survival rate. Collectively, these data demonstrate the preclinical efficacy of targeting the downstream HDAC1/2-Gli1 acetylation in the treatment of SHH-MB.

Dysregulation of the activity of lysine acetyltransferases (KATs) is related to a variety of diseases and/or pathological cellular states; however, their role remains unclear. Therefore, the development of selective modulators of these enzymes is of paramount importance, because these molecules could be invaluable tools for assessing the importance of KATs in several pathologies. We recently found that diethyl pentadecylidenemalonate (SPV106) possesses a previously unobserved inhibitor/activator activity profile against protein acetyltransferases. Herein, we report that manipulation of the carbonyl functions of a series of analogues of SPV106 yielded different activity profiles against KAT2B and KAT3B (pure KAT2B activator, pan-inhibitor, or mixed KAT2B activator/KAT3B inhibitor). Among the novel compounds, a few derivatives may be useful chemical tools for studying the mechanism of lysine acetylation and its implications in physiological and/or pathological processes.

Histone demethylase KDM1A (also known as LSD1) has become an attractive therapeutic target for the treatment of cancer as well as other disorders such as viral infections. We report on the synthesis of compounds derived from the expansion of tranylcypromine as a chemical scaffold for the design of novel demethylase inhibitors. These compounds, which are substituted on the cyclopropyl core moiety, were evaluated for their ability to inhibit KDM1A in vitro as well as to function in cells by modulating the expression of Gfi-1b, a well recognized KDM1A target gene. The molecules were all found to covalently inhibit KDM1A and to become increasingly selective against human monoamine oxidases MAO A and MAO B through the introduction of bulkier substituents on the cyclopropylamine ring. Structural and biochemical analysis of selected trans isomers showed that the two stereoisomers are endowed with similar inhibitory activities against KDM1A, but form different covalent adducts with the FAD co-enzyme.

A comparison of the effects of the 6-(2-chloro-6-fluorobenzyl)-2-(alkylthio)pyrimidin-4(3H)-ones (2-Cl-6-F-S-DABOs) 7-12 and the related 6-(2,6-difluorobenzyl) counterparts 13-15 in HIV-1 infected cells and in the HIV-1 reverse transcriptase (RT) assays is here described. The new 2-Cl-6-F-S-DABOs showed up to picomolar activity against wt HIV-1. Against clinically relevant HIV-1 mutants and in enzyme assays, the simultaneous C5(methyl)/C6(methyl/ethyl) substitution in the 2-Cl-6-F- and 2,6-F2-benzyl series furnished compounds with the highest, wide-spectrum inhibitory activity against HIV-1. Three representative 2-Cl-6-F-S-DABOs carrying two (9c, 10c) or one (10a) stereogenic centers were resolved into their individual stereoisomers and showed a significant diastereo- and enantioselectivity in HIV-1 inhibition, the highest antiviral activity well correlating with the R absolute configuration to the stereogenic center of the C6-benzylic position in both cellular and enzymatic tests. Application of previously reported COMBINEr protocol on 9c and 10c confirmed the influence of the stereogenic centers on their binding modes in the HIV-1 RT.

Recently we described some (thiazol-2-yl)hydrazones as antiprotozoal, antifungal and anti-MAO agents as well as Gcn5 HAT inhibitors. Among these last compounds, CPTH2 and CPTH6 showed HAT inhibition in cells and broad anticancer properties. With the aim to identify HAT inhibitors more potent than the two prototypes, we synthesized several new (thiazol-2-yl)hydrazones including some related thiazolidines and pyrimidin-4(3H)-ones, and we tested the whole library existing in our lab against human p300 and PCAF HAT enzymes. Some compounds (1x, 1c', 1d', 1i' and 2m) were more efficient than CPTH2 and CPTH6 in inhibiting the p300 HAT enzyme. When tested in human leukemia U937 and colon carcinoma HCT116 cells (100 mu M, 30 h), 1x, 1i' and 2m gave higher (U937 cells) or similar (HCT116 cells) apoptosis than CPTH6, and were more potent than CPTH6 in inducing cytodifferentiation (U937 cells). (C) 2014 Elsevier Masson SAS. All rights reserved.

This study investigates the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of H3K9Ac and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5-related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-? general control of amino acid synthesis (GCN5a), determining a relative decrease in total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients. © 2014 by the American Diabetes Association.

Cell division cycle phosphatases CDC25 A, B and C are involved in modulating cell cycle processes and are found overexpressed in a large panel of cancer typology. Here, we describe the development of two novel quinone-polycycle series of CDC25A and C inhibitors on the one hand 1a-k, coumarin-based, and on the other 2a-g, quinolinone-based, which inhibit either enzymes up to a sub-micro molar level and at single-digit micro molar concentrations, respectively. When tested in six different cancer cell lines, compound 2c displayed the highest efficacy to arrest cell viability, showing in almost all cell lines sub-micro molar IC50 values, a profile even better than the reference compound NCS95397. To investigate the putative binding mode of the inhibitors and to develop quantitative structure-activity relationships, molecular docking and 3-D QSAR studies were also carried out. Four selected inhibitors, 1a, 1d, 2a and 2c have been also tested in A431 cancer cells; among them, compound 2c was the most potent one leading to cell proliferation arrest and decreased CDC25C protein levels together with its splicing variant. Compound 2c displayed increased phosphorylation levels of histone H3, induction of PARP and caspase 3 cleavage, highlighting its contribution to cell death through pro-apoptotic effects.

Estrogen and hypoxia promote an aggressive phenotype in prostate cancer (PCa), driving transcription of progression-associated genes. Here, we molecularly dissect the contribution of long non-coding RNA H19 to PCa metastatic potential under combined stimuli, a topic largely uncovered. The effects of estrogen and hypoxia on H19 and cell adhesion molecules&rsquo, expression were investigated in PCa cells and PCa-derived organotypic slice cultures (OSCs) by qPCR and Western blot. The molecular mechanism was addressed by chromatin immunoprecipitations, overexpression, and silencing assays. PCa cells&rsquo, metastatic potential was analyzed by in vitro cell-cell adhesion, motility test, and trans-well invasion assay. We found that combined treatment caused a significant H19 down-regulation as compared with hypoxia. In turn, H19 acts as a transcriptional repressor of cell adhesion molecules, as revealed by up-regulation of both &beta, 3 and &beta, 4 integrins and E-cadherin upon H19 silencing or combined treatment. Importantly, H19 down-regulation and &beta, integrins induction were also observed in treated OSCs. Combined treatment increased both cell motility and invasion of PCa cells. Lastly, reduction of &beta, integrins and invasion was achieved through epigenetic modulation of H19-dependent transcription. Our study revealed that estrogen and hypoxia transcriptionally regulate, via H19, cell adhesion molecules redirecting metastatic dissemination from EMT to a &beta, integrin-mediated invasion.

Introduction: Despite the recent advancement in treating melanoma, options are still limited for patients without BRAF mutations or in relapse from current treatments, and promising, immunotherapies are not without caveats. Thus, alternative options are still in need. Bcl-2 protein is the main member of a family whose best-characterized function is to regulate apoptosis in several pathologies, including melanoma. To evade apoptotic checkpoints, melanoma cells often overexpress Bcl-2 and other antiapoptotic proteins. Consequently, along with the fact that antiapoptotic proteins play a critical role in the efficacy of most chemotherapeutics and play a relevant role in melanoma progression, they have emerged as attractive targets for therapeutic development. Structure-based efforts led to the development of ABT-199 (also known as venetoclax), recently approved by the Food and Drug Administration for the treatment of chronic lymphocytic leukaemia patients. Experimental procedures: To identify new possible selective inhibitors towards antiapoptotic proteins belonging to the bcl-2 family, a computational medicinal chemistry protocol was set up. Both the PubMed/ChEMBL databases, containing known ligands for bcl-2, bcl-w and bcl-xL proteins and their associated affinities, and the RCSB Protein Data Bank listing 65 bcl-2 family protein structures, were used to build preliminary Quantitative Structure-Activity Relationships (QSAR) models. The QSAR models were used to screen the VITAS-M Laboratory database to select 60 chemical scaffolds as hypothetical novel Bcl-2 inhibitors. Surface Plasmon Experiments were carried out to assess affinity of compounds for Bcl-2. The sensitivity of human leukemia and melanoma cells to these compounds was evaluated in terms of cell survival, cell cycle distribution and apoptosis induction, by using different concentrations and time exposure. Results: 9 out of 60 compounds (15%) were able to reduce cell viability at concentrations lower than 50µM, and to induce cell death in a dose-dependent manner, even though at different extend depending on the compound tested. Inclusion of the 60 selected compounds led to a new QSAR model to perform a second virtual screen on the NIH database from which 40 putative Bcl-2 target molecules were selected and whose efficacy to inhibit cell viability of both leukemia and melanoma cells was measured. 11 compounds are endowed with high affinity for Bcl-2, 2 out of 40 compounds were demonstrated to induce apoptosis in both tumor histotypes in a dose-dependent manner, while they did not affect cell viability of human normal fibroblasts. Conclusion: Machine learning application coupled with experimental data have provided new insights into bcl-2 family's regulation of the apoptotic pathway both in leukemia and in melanoma, and suggest that inhibiting the major anti-apoptotic proteins is sufficient to induce cell death. Note: This abstract was not presented at the meeting. Citation Format: Elisabetta Valentini, Marta Di Martile, Marianna Desideri, Simona Carcasole, Alexandros Patsilinakos, Manuela Sabatino, Antonello Mai, Gianni Colotti, Rino Ragno, Daniela Trisciuoglio, Donalella Del Bufalo. Targeting anti-apoptotic Bcl-2 family for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2502.

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

Recent statistics indicate that the human population is ageing rapidly. Healthy, but also diseased, elderly people are increasing. This trend is particularly evident in Western countries, where healthier living conditions and better cures are available. To understand the process leading to age-associated alterations is, therefore, of the highest relevance for the development of new treatments for age-associated diseases, such as cancer, diabetes, Alzheimer and cardiovascular accidents. Mechanistically, it is well accepted that the accumulation of intracellular damage determined by reactive oxygen species (ROS) might orchestrate the progressive loss of control over biological homeostasis and the functional impairment typical of aged tissues. Here, we review how epigenetics takes part in the control of stress stimuli and the mechanisms of ageing physiology and physiopathology. Alteration of epigenetic enzyme activity, histone modifications and DNA-methylation is, in fact, typically associated with the ageing process. Specifically, ageing presents peculiar epigenetic markers that, taken altogether, form the still ill-defined “ageing epigenome”. The comprehension of mechanisms and pathways leading to epigenetic modifications associated with ageing may help the development of anti-ageing therapies.

Histone deacetylase inhibitors (DIs) are promising drugs for the treatment of several pathologies including ischemic and failing heart where they demonstrated efficacy. However, adverse side effects and cardiotoxicity have also been reported. Remarkably, no information is available about the effect of DIs during tissue regeneration following acute peripheral ischemia. In this study, mice made ischemic by femoral artery excision were injected with the DIs MS275 and MC1568, selective for class I and IIa histone deacetylases (HDACs), respectively. In untreated mice, soon after damage, class IIa HDAC phosphorylation and nuclear export occurred, paralleled by dystrophin and neuronal nitric-oxide synthase (nNOS) down-regulation and decreased protein phosphatase 2A activity. Between 14 and 21 days after ischemia, dystrophin and nNOS levels recovered, and class IIa HDACs relocalized to the nucleus. In this condition, the MC1568 compound increased the number of newly formed muscle fibers but delayed their terminal differentiation, whereas MS275 abolished the early onset of the regeneration process determining atrophy and fibrosis. The selective DIs had differential effects on the vascular compartment: MC1568 increased arteriogenesis whereas MS275 inhibited it. Capillarogenesis did not change. Chromatin immunoprecipitations revealed that class IIa HDAC complexes bind promoters of proliferation-associated genes and of class I HDAC1 and 2, highlighting a hierarchical control between class II and I HDACs during tissue regeneration. Our findings indicate that class-selective DIs interfere with normal mouse ischemic hindlimb regeneration and suggest that their use could be limited by alteration of the regeneration process in peripheral ischemic tissues.

The reactivity of 4-(1-butoxyvinyl)-2H-chromen-2-one (1) and (E)-4-(2-butoxyvinyl)-2H-chromen-2-one (2) as diene in thermal Diels–Alder cycloaddition reactions with several electron-poor dienophiles is reported. Among several dienophiles used in this study 1,4-benzoquinone afforded cycloadducts 11-butoxy-1H-naphtho[1,2-c]chromene-1,4,5-trione (3e) and 1H-naphtho[1,2-c]chromene-1,4,5-trione (4g) that showed Cdc25 phosphatase-inhibition activity at low micromolar values, with both compounds more effective against Cdc25 A and Cdc25 C isoforms.

Chromatin modifications are sensitive to environmental and nutritional stimuli. Abnormalities in epigenetic regulation are associated with metabolic disorders such as obesity and diabetes that are often linked with defects in oxidative metabolism. Here, we evaluated the potential of class-specific synthetic inhibitors of histone deacetylases (HDACs), central chromatin-remodeling enzymes, to ameliorate metabolic dysfunction. Cultured myotubes and primary brown adipocytes treated with a class I–specific HDAC inhibitor showed higher expression of Pgc-1α, increased mitochondrial biogenesis, and augmented oxygen consumption. Treatment of obese diabetic mice with a class I– but not a class II–selective HDAC inhibitor enhanced oxidative metabolism in skeletal muscle and adipose tissue and promoted energy expenditure, thus reducing body weight and glucose and insulin levels. These effects can be ascribed to increased Pgc-1α action in skeletal muscle and enhanced PPARγ/PGC-1α signaling in adipose tissue. In vivo ChIP experiments indicated that inhibition of HDAC3 may account for the beneficial effect of the class I–selective HDAC inhibitor. These results suggest that class I HDAC inhibitors may provide a pharmacologic approach to treating type 2 diabetes.

International audience; Chemical manipulations performed on the histone H3 lysine 9 methyltransferases (G9a/GLP) inhibitor BIX-01294 afforded novel desmethoxyquinazolines able to inhibit the DNA methyltransferase DNMT3A at low micromolar levels without any significant inhibition of DNMT1 and G9a. In KG-1 cells such compounds, when tested at sub-toxic doses, induced the luciferase re-expression in a stable construct controlled by a cytomegalovirus (CMV) promoter silenced by methylation (CMV-luc assay). Finally, in human lymphoma U-937 and RAJI cells, the N-(1-benzylpiperidin-4-yl)-2-(4-phenylpiperazin-1-yl)quinazolin-4-amine induced the highest proliferation arrest and cell death induction starting from 10 µM, in agreement with its DNMT3A inhibitory potency.

SETD8/SET8/Pr-SET7/KMT5A is the only known lysine methyltransferase (KMT) that monomethylates lysine 20 of histone H4 (H4K20) in vivo. Lysine residues of non-histone proteins including proliferating cell nuclear antigen (PCNA) and p53 are also monomethylated. As a consequence, the methyltransferase activity of the enzyme is implicated in many essential cellular processes including DNA replication, DNA damage response, transcription modulation, and cell cycle regulation. This review aims to provide an overview of the roles of SETD8 in physiological and pathological pathways and to discuss the progress made to date in inhibiting the activity of SETD8 by small molecules, with an emphasis on their discovery, selectivity over other methyltransferases and cellular activity.

Chemical manipulations performed on 2-methyl-3-carbethoxyquinoline (1), a histone acetyltransferase inhibitor previously identified by our research group and active at the sub-millimolar/millimolar level, led to compounds bearing higher alkyl groups at the C2-quinoline or additional side chains at the C6-quinoline positions. Such compounds displayed at least threefold improved inhibitory potency toward p300 protein lysine acetyltransferase activity; some of them decreased histone H3 and H4 acetylation levels in U937 cells and induced high degrees of apoptosis (three compounds >10-fold higher than compound 1) after treatment of U937 cells.

SETD8/SET8/Pr-SET7/KMT5A is the only known lysine methyltransferase that monomethylates lysine 20 of histone H4 (H4K20) in vivo. The methyltransferase activity of SETD8 has been implicated in many essential cellular processes, including DNA replication, DNA damage response, transcription modulation, and cell cycle regulation. In addition to H4K20, SETD8 monomethylates non-histone substrates including proliferating cell nuclear antigen and p53. During the past decade, different structural classes of inhibitors targeting various lysine methyltransferases have been designed and developed. However, the development of SETD8 inhibitors is still in its infancy. This review covers the progress made to date in inhibiting the activity of SETD8 by small molecules, with an emphasis on their discovery, selectivity over other methyltransferases, and cellular activity.

The binding mode of newly discovered histone demethylase inhibitors could have applications in the design and repurposing of drugs., Because of its involvement in the progression of several malignant tumors, the histone lysine-specific demethylase 1 (LSD1) has become a prominent drug target in modern medicinal chemistry research. We report on the discovery of two classes of noncovalent inhibitors displaying unique structural features. The antibiotics polymyxins bind at the entrance of the substrate cleft, where their highly charged cyclic moiety interacts with a cluster of positively charged amino acids. The same site is occupied by quinazoline-based compounds, which were found to inhibit the enzyme through a most peculiar mode because they form a pile of five to seven molecules that obstruct access to the active center. These data significantly indicate unpredictable strategies for the development of epigenetic inhibitors.

Lysine acetylations are reversible posttranslational modifications of histone and non-histone proteins that play important regulatory roles in signal transduction cascades and gene expression. Lysine acetylations are regulated by histone acetyltransferases as writers and histone deacetylases as erasers. Because of their role in signal transduction cascades, these enzymes are important players in inflammation. Therefore, applications of histone acetyltransferase inhibitors to reduce inflammatory responses are interesting. Among the few histone acetyltransferase inhibitors described, C646 is one of the most potent (Ki of 0.4μM for histone acetyltransferase p300). C646 was described to regulate the NF-κB pathway; an important pathway in inflammatory responses, which is regulated by acetylation. Interestingly, this pathway has been implicated in asthma and COPD. Therefore we hypothesized that via regulation of the NF-κB signaling pathway, C646 can inhibit pro-inflammatory gene expression, and have potential for the treatment of inflammatory lung diseases. In line with this, here we demonstrate that C646 reduces pro-inflammatory gene expression in RAW264.7 murine macrophages and murine precision-cut lung slices. To unravel its effects on cellular substrates we applied mass spectrometry and found, counterintuitively, a slight increase in acetylation of histone H3. Based on this finding, and structural features of C646, we presumed inhibitory activity of C646 on histone deacetylases, and indeed found inhibition of histone deacetylases from 7μM and higher concentrations. This indicates that C646 has potential for further development towards applications in the treatment of inflammation, however, its newly discovered lack of selectivity at higher concentrations needs to be taken into account.

Among epigenetic enzymes, histone deacetylases (HDACs) are responsible for regulating the expression of an extensive array of genes by reversible deacetylation of nuclear histones as well as a large number of non-histone proteins. Initially proposed for cancer therapy, recently the interest for HDAC inhibitors (HDACi) as orally active, safe, and anti-inflammatory agents is rising due to their ability in reducing the severity of inflammatory and autoimmune diseases. In particular, selective HDAC3, HDAC6, and HDAC8 inhibitors have been described to downregulate the expression of pro-inflammatory cytokines (TNF-α, TGF-β, IL-1β, and IL-6). Herein, using KB31, C2C12, and 3T3-J2 cell lines, we demonstrated that, under lipopolysaccharide-induced in vitro inflammation, HDAC3/6/8 inhibitor MC2625 and HDAC6-selective inhibitor MC2780 were effective at a concentration of 30 ng/mL to downregulate mRNA expression of pro-inflammatory cytokines (IL-1β and IL-6) and to promote the transcription of IL-10 gene, without affecting the cell viability. Afterwards, we investigated by immunohistochemistry the activity of MC2625 and MC2780 at a concentration of 60 ng/kg animal weight to regulate silicone-triggered immune response in C57BL/6J female mice. Our findings evidenced the ability of such inhibitors to reduce host inflammation in silicone implants promoting a thickness reduction of peri-implant fibrous capsule, upregulating IL-10 expression, and reducing the production of both IL-1β and IL-6. These results underline the potential application of MC2625 and MC2780 in inflammation-related diseases.

Article, Sirtuins are NAD+-dependent histone deacetylases regulating important metabolic pathways in prokaryotes and eukaryotes and are involved in many biological processes such as cell survival, senescence, proliferation, apoptosis, DNA repair, cell metabolism, and caloric restriction. The seven members of this family of enzymes are considered potential targets for the treatment of human pathologies including neurodegenerative diseases, cardiovascular diseases, and cancer. Furthermore, recent interest focusing on sirtuin modulators as epigenetic players in the regulation of fundamental biological pathways has prompted increased efforts to discover new small molecules able to modify sirtuin activity. Here, we review the role, mechanism of action, and biological function of the seven sirtuins, as well as their inhibitors and activators., published version, http://purl.org/eprint/status/PeerReviewed

Modulators of sirtuins are considered promising therapeutic targets for the treatment of cancer, cardiovascular, metabolic, inflammatory, and neurodegenerative diseases. Here we prepared new 1,4-dihydropyridines (DHPs) bearing changes at the C2/C6, C3/C5, C4, or N1 position. Tested with the SIRTainty procedure, some of them displayed increased SIRT1 activation with respect to the prototype 3a, high NO release in HaCat cells, and ameliorated skin repair in a mouse model of wound healing. In C2C12 myoblasts, two of them improved mitochondrial density and functions. All the effects were reverted by coadministration of compound C (9), an AMPK inhibitor, or of EX-527 (10), a SIRT1 inhibitor, highlighting the involvement of the SIRT1/AMPK pathway in the action of DHPs. Finally, tested in a panel of cancer cells, the water-soluble form of 3a, compound 8, displayed antiproliferative effects in the range of 8-35 μM and increased H4K16 deacetylation, suggesting a possible role for SIRT1 activators in cancer therapy.

Lysine demethylase 1 (LSD1) plays an important role in mediating the expression of genes involved in cancer and non-cancer diseases such as viral infections, cardiovascular and neurodegenerative disorders. It is involved in a number of processes from adipogenesis to cell adhesion to viral latency, regulating several cell pathways related with proliferation, development, and cell cycle control. Numerous chemical entities have been studied in recent years and some of them entered the clinical arena.This review summarizes recent efforts in the drug development of LSD1 inhibitors reported in the patent literature covering the 2010-2015 period, including their potential use as therapeutics in cancerous, neurological, inflammatory, cardiovascular, and viral diseases.The development of novel potent and selective LSD1 inhibitors is ongoing, in order to improve their potency and selectivity against specific types of cancer or non-cancer diseases. More in-depth studies are required to assess the role of LSD1 inhibitors in the expression of LSD1 target genes, for a better assessment of the biochemistry underlying their efficacy, and to provide evidence for any possible side effects. Furthermore, an interesting therapeutic approach is the use of LSD1 inhibitors in conjunction with other epidrugs to combine their therapeutic potential, leading to innovative, personalized treatments.

SETD8/SET8/Pr-SET7/KMT5A is the only known lysine methyltransferase that monomethylates lysine 20 of histone H4 (H4K20) in vivo. The methyltransferase activity of SETD8 has been implicated in many essential cellular processes, including DNA replication, DNA damage response, transcription modulation, and cell cycle regulation. In addition to H4K20, SETD8 monomethylates non-histone substrates including proliferating cell nuclear antigen and p53. During the past decade, different structural classes of inhibitors targeting various lysine methyltransferases have been designed and developed. However, the development of SETD8 inhibitors is still in its infancy. This review covers the progress made to date in inhibiting the activity of SETD8 by small molecules, with an emphasis on their discovery, selectivity over other methyltransferases, and cellular activity.

Aim: Histone deacetylases (HDACs) regulate the life cycle of several viruses. We investigated the ability of different HDAC inhibitors, to interfere with influenza virus A/Puerto Rico/8/34/H1N1 (PR8 virus) replication in Madin-Darby canine kidney and NCI cells. Results: 3-(5-(3-Fluorophenyl)-3-oxoprop-1-en-1-yl)-1-methyl-1H-pyrrol-2-yl)-N-hydroxyacrylamide (MC1568) inhibited HDAC6/8 activity and PR8 virus replication, with decreased expression of viral proteins and their mRNAs. Such an effect may be related to a decrease in intranuclear content of viral polymerases and, in turn, to an early acetylation of Hsp90, a major player in their nuclear import. Later, the virus itself induced Hsp90 acetylation, suggesting a differential and time-dependent role of acetylated proteins in virus replication. Conclusion: The inhibition of HDAC6/8 activity during early steps of PR8 virus replication could lead to novel anti-influenza strategy.

Cancer stem cells (CSCs) play an important role in tumor initiation, progression, therapeutic failure and tumor relapse. In this study, we evaluated the efficacy of the thiazole derivative 3-methylcyclopentylidene-[4-(4′-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6), a novel pCAF and Gcn5 histone acetyltransferase inhibitor, as a small molecule that preferentially targets lung cancer stem-like cells (LCSCs) derived from non-small cell lung cancer (NSCLC) patients. Notably, although CPTH6 inhibits the growth of both LCSC and NSCLC cell lines, LCSCs exhibit greater growth inhibition than established NSCLC cells. Growth inhibitory effect of CPTH6 in LCSC lines is primarily due to apoptosis induction. Of note, differentiated progeny of LCSC lines is more resistant to CPTH6 in terms of loss of cell viability and reduction of protein acetylation, when compared to their undifferentiated counterparts. Interestingly, in LCSC lines CPTH6 treatment is also associated with a reduction of stemness markers. By using different HAT inhibitors we provide clear evidence that inhibition of HAT confers a strong preferential inhibitory effect on cell viability of undifferentiated LCSC lines when compared to their differentiated progeny. In vivo, CPTH6 is able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Strikingly, the ability of CPTH6 to inhibit tubulin acetylation is also confirmed in vivo. Overall, our studies propose histone acetyltransferase inhibition as an attractive target for cancer therapy of NSCLC.

It is well established that the activity of chromatin-modifying enzymes is crucial for regulating gene expression associated with hippocampal-dependent memories. However, very little is known about how these epigenetic mechanisms influence the formation of cortically dependent memory, particularly when there is competition between opposing memory traces, such as that which occurs during the acquisition and extinction of conditioned fear. Here we demonstrate, in C57BL/6 mice, that the activity of p300/CBP-associated factor (PCAF) within the infralimbic prefrontal cortex is required for long-term potentiation and is necessary for the formation of memory associated with fear extinction, but not for fear acquisition. Further, systemic administration of the PCAF activator SPV106 enhances memory for fear extinction and prevents fear renewal. The selective influence of PCAF on fear extinction is mediated, in part, by a transient recruitment of the repressive transcription factor ATF4 to the promoter of the immediate early genezif268, which competitively inhibits its expression. Thus, within the context of fear extinction, PCAF functions as a transcriptional coactivator, which may facilitate the formation of memory for fear extinction by interfering with reconsolidation of the original memory trace.

BIX-01294 and its analogs were originally identified and subsequently designed as potent inhibitors against histone H3 lysine 9 (H3K9) methyltransferases G9a and G9a-like protein. Here, we show that BIX-01294 and its analog E67 can also inhibit H3K9 Jumonji demethylase KIAA1718 with half-maximal inhibitory concentrations in low micromolar range. Crystallographic analysis of KIAA1718 Jumonji domain in complex with E67 indicated that the benzylated six-membered piperidine ring was disordered and exposed to solvent. Removing the moiety (generating compound E67-2) has no effect on the potency against KIAA1718 but, unexpectedly, lost inhibition against G9a-like protein by a factor of 1500. Furthermore, E67 and E67-2 have no effect on the activity against histone H3 lysine 4 (H3K4) demethylase JARID1C. Thus, our study provides a new avenue for designing and improving the potency and selectivity of inhibitors against H3K9 Jumonji demethylases over H3K9 methyltransferases and H3K4 demethylases.

In the past years, a lot of attention has been given to the identification and characterization of selective and potent inhibitors of chromatin-modifying enzymes to better understand their specific role in transcriptional regulation. As aberrant histone methylation is involved in different pathological processes, the search for methyltransferase and demethylase inhibitors has emerged as a crucial issue in current medicinal chemistry research. High-throughput in vitro assays are important tools for the identification of new methyltransferase or demethylase inhibitors. These usually use oligopeptide substrates derived from histone sequences, although in many cases, they are not good substrates for these enzymes. Here, the authors report about the setup and establishment of in vitro assays that use native core histones as substrates, enabling an assay environment that better resembles native conditions. They have applied these substrates for the known formaldehyde dehydrogenase assay for the histone demethylase LSD1 and have established two new antibody-based assays. For LSD1, a heterogeneous assay format was set up, and a homogeneous assay was used for the characterization of the arginine methyltransferase PRMT1. Validation of the system was achieved with reference inhibitors in each case.

Monoamine oxidases (MAOs) are involved in various psychiatric and neurodegenerative disorders; hence, MAO inhibitors are useful agents in the therapy of Parkinson's disease, Alzheimer's dementia, and depression syndrome. Herein we report a novel series of 3-(1H-pyrrol-3-yl)-2-oxazolidinones 3-7 as reversible, highly potent and selective anti-MAO-A agents. In particular, 4b, 5b, and 4c showed a K(i-MAO-A) of 0.6, 0.8, and 1 nM, respectively, 4c being 200000-fold selective for MAO-A with respect to MAO-B.

OBJECTIVE Class IIa histone deacetylases (HDACs) belong to a large family of enzymes involved in protein deacetylation and play a role in regulating gene expression and cell differentiation. Previously, we showed that HDAC inhibitors modify the timing and determination of pancreatic cell fate. The aim of this study was to determine the role of class IIa HDACs in pancreas development. RESEARCH DESIGN AND METHODS We took a genetic approach and analyzed the pancreatic phenotype of mice lacking HDAC4, -5, and -9. We also developed a novel method of lentiviral infection of pancreatic explants and performed gain-of-function experiments. RESULTS We show that class IIa HDAC4, -5, and -9 have an unexpected restricted expression in the endocrine β- and δ-cells of the pancreas. Analyses of the pancreas of class IIa HDAC mutant mice revealed an increased pool of insulin-producing β-cells in Hdac5−/− and Hdac9−/− mice and an increased pool of somatostatin-producing δ-cells in Hdac4−/− and Hdac5−/− mice. Conversely, HDAC4 and HDAC5 overexpression showed a decreased pool of insulin-producing β-cells and somatostatin-producing δ-cells. Finally, treatment of pancreatic explants with the selective class IIa HDAC inhibitor MC1568 enhances expression of Pax4, a key factor required for proper β-and δ-cell differentiation and amplifies endocrine β- and δ-cells. CONCLUSIONS We conclude that HDAC4, -5, and -9 are key regulators to control the pancreatic β/δ-cell lineage. These results highlight the epigenetic mechanisms underlying the regulation of endocrine cell development and suggest new strategies for β-cell differentiation-based therapies.

The transcriptional peroxisome proliferator-activated receptor γ (PPARγ) co-activator PGC-1α plays a central role in the regulation of cellular energy metabolism. Among the wide range of its activities, PGC-1α controls mitochondrial biogenesis and function and is one of the main factors involved in hormonal and nutrient regulation of hepatic gluconeogenesis. PGC-1α is present in a multiprotein complex, and its activity can also be modulated through epigenetic modifications. In particular, it is directly acetylated by the HAT enzyme general control nonderepressible 5 (GCN5), resulting in a transcriptionally inactive protein that relocalizes from promoter regions to nuclear foci, whereas it is deacetylated by SIRT1 at multiple lysine sites, with a subsequent increase in its activity leading to induction of liver gluconeogenic gene transcription. Thus, both GCN5 and SIRT1 may be pharmacological targets to regulate the activity of PGC-1α, providing a potential treatment for metabolic disorders in which hepati...

"In this report we describe the synthesis and biological characterization of two series of sirtuins' inhibitors (SIRTi), designed as simplification products of the previously reported SIRT1-selective inhibitor MC2141 (4). In the first series (5a-t) we report a number of 2-substituted-1,2-dihydrobenzo[f]chromen-3-ones with a marked selectivity for the inhibition of SIRT2 over SIRT1. Some of such derivatives showed also high pro-apoptotic (5i and 5l) and\/or cytodifferentiating (5d, 5i, and 5o) properties in a human leukemia cell line (U937). The second group of SIRTi (6a-q) is characterized by some analogues of cambinol (3), a well known SIRTi active against the Burkitt lymphoma. Such compounds, differently from the unselective prototype, are endowed with a selective inhibition of SIRT1 over SIRT2, and, in some cases (6j, 6k, and 6q), are more efficient than 3 to induce apoptosis in U937 cells."

Summary2-oxoglutarate (2-OG)-dependent oxygenases have diverse roles in human biology. The inhibition of several 2-OG oxygenases is being targeted for therapeutic intervention, including for cancer, anemia, and ischemic diseases. We report a small-molecule probe for 2-OG oxygenases that employs a hydroxyquinoline template coupled to a photoactivable crosslinking group and an affinity-purification tag. Following studies with recombinant proteins, the probe was shown to crosslink to 2-OG oxygenases in human crude cell extracts, including to proteins at endogenous levels. This approach is useful for inhibitor profiling, as demonstrated by crosslinking to the histone demethylase FBXL11 (KDM2A) in HEK293T nuclear extracts. The results also suggest that small-molecule probes may be suitable for substrate identification studies.

New spiro[chromane-2,4'-piperidine] and spiro[benzofuran-2,4'-piperidine] hydroxamic acid derivatives as HDAC inhibitors have been identified by combining privileged structures with a hydroxamic acid moiety as zinc binding group. The compounds were evaluated for their ability to inhibit nuclear extract HDACs and for their in vitro antiproliferative activity on different tumor cell lines. This work resulted in the discovery of spirocycle 30d that shows good oral bioavailability and tumor growth inhibition in an HCT-116 murine xenograft model.

Here, we describe a novel small series of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that combine peculiar structural features of diarylpyrimidines (DAPYs) and dihydro-alkoxy-benzyl-oxopyrimidines (DABOs). These DAPY-DABO hybrids (1-4) showed a characteristic SAR profile and a nanomolar anti-HIV-1 activity at both enzymatic and cellular level. In particular, the two compounds 4d and 2d, with a (sub)nanomolar activity against wild-type and clinically relevant HIV-1 mutant strains, were selected as lead compounds for next optimization studies.

This Special Issue of ChemMedChem on Epigenetics and Drug Discovery brings together a diverse group of papers on a range of topics from bromodomain inhibitors and long noncoding RNAs to classic agents targeting histone deacetylases (HDACs) and less typical HDAC inhibitors, such as the chimeric compounds designed by Greg Thatcher et al. and featured on the Front Cover of the issue that both target HDACs and act as selective estrogen receptor modulators (SERMs).

Epigenetic deregulation contributes to diseases including cancer, neurodegeneration, osteodystrophy, cardiovascular defects, and obesity. For this reason, several inhibitors for histone deacetylases (HDACs) are being validated as novel anti-cancer drugs in clinical studies and display important anti-proliferative activities. While most inhibitors act on both class I, II, and IV HDACs, evidence is accumulating that class I is directly involved in regulation of cell growth and death, whereas class II members regulate differentiation processes, such as muscle and neuronal differentiation. Here, we show that the novel class II-selective inhibitor MC1568 interferes with the RAR- and peroxisome proliferator-activated receptor γ (PPARγ)-mediated differentiation-inducing signaling pathways. In F9 cells, this inhibitor specifically blocks endodermal differentiation despite not affecting retinoic acid-induced maturation of promyelocytic NB4 cells. In 3T3-L1 cells, MC1568 attenuates PPARγ-induced adipogenesis, while the class I-selective MS275 blocked adipogenesis completely thus revealing a different mode of action and/or target profile of the two classes of HDACs. Using in vivo reporting PPRE-Luc mice, we find that MC1568 impairs PPARγ signaling mostly in the heart and adipose tissues. These results illustrate how HDAC functions can be dissected by selective inhibitors.

A series of amidopropenyl hydroxamic acid derivatives were prepared as novel inhibitors of human histone deacetylases (HDACs). Several compounds showed potency at

The alkylation of 5-alkyl-6-(2,6-dihalobenzyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-ones with MeI, AllSCH2Cl, and MeSCH2Cl in the K2CO3–DMF, NaOMe–MeOH, and KOH–EtOH systems was investigated. A hypothetical mechanism for the reaction is examined, and an explanation is proposed for the composition of the reaction products. The presence of high anti-HIV-1 activity was established in the obtained derivatives of 2-{[(allylsulfanyl)methyl]sulfanyl}pyrimidin-4(3H)-one.