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Silencing of DNMTs inhibits cell proliferation, induces osteoblastic differentiation and enhances DXR sensitivity

Inhibitory activity of MC3343 against human DNMT1 and human DNMT3a

The identification of new therapeutic strategies against osteosarcoma, the most common primary bone tumor, continues to be a primary goal to improve the outcomes of patients refractory to conventional chemotherapy. Osteosarcoma originates from the transformation of mesenchymal stem cells (MSC) and/or osteoblast progenitors, and the loss of differentiation is a common biological osteosarcoma feature, which has strong significance in predicting tumor aggressiveness. Thus, restoring differentiation through epigenetic reprogramming is potentially exploitable for therapeutic benefits. Here, we demonstrated that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 affected tumor proliferation by blocking osteosarcoma cells in G1 or G2–M phases and induced osteoblastic differentiation through the specific reexpression of genes regulating this physiologic process. Although MC3343 has a similar antiproliferative effect as 5azadC, the conventional FDA-approved nucleoside inhibitor of DNA methylation, its effects on cell differentiation are distinct. Induction of the mature osteoblast phenotype coupled with a sustained cytostatic response was also confirmed in vivo when MC3343 was used against a patient-derived xenograft (PDX). In addition, MC3343 displayed synergistic effects with doxorubicin and cisplatin (CDDP), two major chemotherapeutic agents used to treat osteosarcoma. Specifically, MC3343 increased stable doxorubicin bonds to DNA, and combined treatment resulted in sustained DNA damage and increased cell death. Overall, this nonnucleoside DNMTi is an effective novel agent and is thus a potential therapeutic option for patients with osteosarcoma who respond poorly to preadjuvant chemotherapy. Mol Cancer Ther; 17(9); 1881–92. ©2018 AACR.

Schistosoma mansoni HDAC8 is a reliable target to fight schistosomiasis, and several inhibitors have been reported in the literature up to now. Nevertheless, only a few displayed selectivity over the human deacetylases and some exhibited very low or no activity against parasite larvae and/or adult worms. We report here the in vitro enzyme and biological activity of a small library of HDAC inhibitors from our lab, in many cases exhibiting submicromolar/nanomolar potency against smHDAC8 and diverse degrees of selectivity over hHDAC1 and/or hHDAC6. Such compounds were tested against schistosomula, and a selection of them against the adult forms of S. mansoni, to detect their effect on viability. Some of them showed the highest viability reduction for the larval stage with IC

Neglected tropical diseases (NTDs), including trypanosomiasis, leishmaniasis, and schistosomiasis, result in a significant burden in terms of morbidity and mortality worldwide every year. Current antiparasitic drugs suffer from several limitations such as toxicity, no efficacy toward all of the forms of the parasites' life cycle, and/or induction of resistance. Histone-modifying enzymes play a crucial role in parasite growth and survival; thus, the use of epigenetic drugs has been suggested as a strategy for the treatment of NTDs. We tested structurally different HDACi

Programmed death-ligand 1 (PD-L1) is an immune checkpoint protein the overexpression of which results in an inhibitory signal that induces T cell exhaustion responsible for immune escape in tumors. Immunotherapy strategies targeting the PD-L1 pathway have achieved remarkable success in treating various types of cancer. More recently, numerous advances in understanding the complex PD-L1 biology have been made, and the first small-molecule inhibitors have been described in the literature. In this review, we highlight the most promising recent advances in understanding the complex regulation mechanisms focusing on small-molecule modulators, which could be used in rational therapy combinations with other epigenetic chemotherapeutic agents.

LSD1 is a lysine demethylase highly involved in initiation and development of cancer. To design highly effective covalent inhibitors, a strategy is to fill its large catalytic cleft by designing tranylcypromine (TCP) analogs decorated with long, hindered substituents. We prepared three series of TCP analogs, carrying aroyl- and arylacetylamino (1 a-h), Z-amino acylamino (2 a-o), or double-substituted benzamide (3 a-n) residues at the C4 or C3 position of the phenyl ring. Further fragments obtained by chemical manipulation applied on the TCP scaffold (compounds 4 a-i) were also prepared. When tested against LSD1, most of 1 and 3 exhibited IC50 values in the low nanomolar range, with 1 e and 3 a,d,f,g being also the most selective respect to monoamine oxidases. In MV4-11 AML and NB4 APL cells compounds 3 were the most potent, displaying up to sub-micromolar cell growth inhibition against both cell lines (3 a) or against NB4 cells (3 c). The most potent compounds in cellular assays were also able to induce the expression of LSD1 target genes, such as GFI-1b, ITGAM, and KCTD12, as functional read-out for LSD1 inhibition. Mouse and human intrinsic clearance data highlighted the high metabolic stability of compounds 3 a, 3 d and 3 g. Further studies will be performed on the new compounds 3 a and 3 c to assess their anticancer potential in different cancer contexts.

Lymphomas are still difficult to treat even with modern therapies as, among others, multidrug resistance (MDR) is often counteracting a successful cancer therapy. P-gp/ABC-transporters are well-known for their crucial role in the main tumour MDR mechanism, eliminating drugs and cytotoxic substances from the cancer cell by efflux, and their modulators are promising for innovative therapy, but none has been approved in the pharmaceutical market yet. Herein, we have designed, synthesised and analysed 30 novel seleno- and thioether 1,3,5-triazine derivatives conducting comprehensive studies to evaluate their potential application in human JURKAT lymphoma cells. Among the new compounds, four (11, 12, 13 and 23) were much more effective than the reference inhibitor verapamil, being potent ABCB1 inhibitors already at 2 μM, while 5 and 15 showed very potent ABCB1 inhibitory activity only at 20 μM. Results of P-gp ATPase assays, supported with docking studies, indicated the competitive substrate mode of modulating action for 15, while ABCB1, ABCC1 and ABCG2 genes expression induction by 15 with q-PCR was confirmed. All compounds were evaluated for their cytotoxic and antiproliferative properties in both sensitive (PAR) and resistant (MDR) mouse T-lymphoma cell lines, and compound 15, also considering its promising ABCB1 inhibition properties, was revealed to be the best compound in terms of its cytotoxic effect (IC

In recent years there has been an increase in the development of new synthetic drugs, among which the "bath salt" 3,4-methylenedioxypyrovalerone (MDPV), a psychostimulant with a mechanism of action similar to those of cocaine and amphetamine, stands out. Drugs of abuse have been consistently shown to affect memory function in male rodents. We have recently shown that amphetamine and MDPV induce generalization of fear memory in an inhibitory avoidance discrimination task in male rats. Although abuse of illicit drugs is more prevalent in men than in women, several studies have demonstrated that females are more vulnerable to the effects of drugs of abuse than males and the effects caused by substance dependence on memory in females are still under-investigated. Thus, we examined the effects of subchronic amphetamine or MDPV administrations on memory in a contextual fear conditioning/generalization paradigm in adult male and female rats. Animals were given daily subchronic injections of the drugs, starting 6 days prior to the beginning of the behavioral procedures until the end of the paradigm. On day 1 of the experimental protocol, all rats were exposed to a safe context and, the day after, to a slightly different chamber where they received an unsignaled footshock. Twenty-four and forty-eight hours later, freezing behavior and emission of 22 kHz-ultrasonic vocalizations (USVs) were measured in the two different contexts to assess fear memory retention and generalization. Our results indicate that MDPV treatment altered freezing in both sexes, USVs were affected by amphetamine in males while by MDPV in females.

Background: The substantial increase in use of 3,4-methylenedioxypyrovalerone (MDPV), a popular recreational synthetic cathinone, has raised legitimate questions about its behavioral consequences and abuse liability. Aims: The aim of this study was to study MDPV-induced neurobehavioral effects in the rat, using different paradigms traditionally developed to study drug-attributed addictive properties. Methods: Different patterns of intraperitoneal 3 mg/kg MDPV administration were investigated. Consequences on rat horizontal locomotion and behavior of acute, intermittent (once daily dosing over 10 days), and binge (three-time daily dosing for 3 days) MDPV administration as well as challenge after 10 day MDPV withdrawal were studied. The dopamine receptor-D1 antagonist, SCH23390, was bilaterally infused in the nucleus accumbens to determine the role of D1-receptors in MDPV-related effects on the associative memory recall using the conditioned place preference paradigm. In addition, in a separate experience using western blot, we investigated the effects of chronic MDPV administration (four injections during 24 h) on ΔFosB expression in the nucleus accumbens, caudate putamen, and prefrontal cortex. Results: Acute MDPV administration increased stereotypies and open arm entries in the elevated plus maze while SCH23390 abolished MDPV-induced enhancing effects on memory consolidation. Intermittent MDPV administration resulted in sensitization of MDPV-induced locomotor effects and tolerance during the following challenge. With binge MDPV administration, locomotor activity was not altered despite tolerance onset after challenge. SCH23390 abolished MDPV-induced conditioned place preference. Chronic MDPV administration induced ΔFosB accumulation in the nucleus accumbens, caudate putamen, and prefrontal cortex. Conclusions: Our findings clearly show that MDPV produces profound behavioral alterations mediated by the activation of the dopaminergic system similarly to other amphetamines.

Disruption of EZH2-embryonic ectoderm development (EED) protein-protein interaction (PPI) is a new promising cancer therapeutic strategy. We have previously reported the discovery of astemizole, a small-molecule inhibitor targeting the EZH2-EED PPI. Herein, we report the cocrystal structure of EED in complex with astemizole at 2.15 A. The structure elucidates the detailed binding mode of astemizole to EED and provides a structure-guided design for the discovery of a novel EZH2-EED interaction inhibitor, DC-PRC2in-01, with an affinity Kd of 4.56 μM. DC-PRC2in-01 destabilizes the PRC2 complex, thereby leading to the degradation of PRC2 core proteins and the decrease of global H3K27me3 levels in cancer cells. The proliferation of PRC2-driven lymphomas cells is effectively inhibited, and the cell cycle is arrested in the G0/G1 phase. Together, these data demonstrate that DC-PRC2in-01 could be an effective chemical probe for investigating the PRC2-related physiology and pathology and providing a promising chemical scaffold for further development.

Epigenetics is nowadays a well-accepted area of research. In the last years, tremendous progress was made regarding molecules targeting EZH2, directly or indirectly. Recently tazemetostat hit the market after FDA-approval for the treatment of lymphoma. However, the impairment of EZH2 activity by orthosteric intervention has proven to be effective only in a limited subset of cancers. Considering the multiproteic nature of the PRC2 complex and the marked dependence of EZH2 functions on the other core subunits such as EED, in recent years, a new targeting approach ascended to prominence. The possibility to cripple the function of the PRC2 complex by interfering with its multimeric integrity fueled the interest in developing EZH2-EED protein-protein interaction and EED inhibitors as indirect modulators of PRC2-dependent methyltransferase activity. In this Perspective, we aim to summarize the latest findings regarding the development and the biological activity of these emerging classes of PRC2 modulators from a medicinal chemist's viewpoint.

m6A is emerging as one of the most important RNA modifications because of its involvement in pathological and physiological events. Here, we provide an overview of this epitranscriptomic modification, beginning with a description of the molecular players involved and continuing with a focus on the role of m6A in the maintenance of stemness, induction of the epithelial to mesenchymal transition (EMT), and tumor progression. Finally, we discuss the state of the art regarding the design and validation of inhibitors of m6A writers or erasers to provide a background for future investigations and for the development of specific therapeutics.

Selenium-containing compounds have emerged as a potentially promising treatment for viral infections and tumor development and dissemination. Selenium per se is often considered as a toxic element with little or no beneficial effects, but considerable advances have been made in the understanding of the complex biology, chemistry and drug delivery of this element, especially when it is included in bioactive molecules. Here, we summarize and critically discuss recent findings in the field of selenium-based applications for the treatment of cancer and viral infections.

Starting from the N-hydroxy-3-(4-(2-phenylbutanoyl)amino)phenyl)acrylamide (5 b) previously described by us as a HDAC inhibitor, we prepared four aza-analogues, 6-8, 9 b, as regioisomers containing the pyridine nucleus. Preliminary screening against mHDAC1 highlighted the N-hydroxy-5-(2-(2-phenylbutanoyl)amino)pyridyl)acrylamide (9 b) as the most potent inhibitor. Thus, we further developed both pyridylacrylic- and nicotinic-based hydroxamates (9 a, 9 c-f, and 11 a-f) and 2'-aminoanilides (10 a-f and 12 a-f), related to 9 b, to be tested against HDACs. Among them, the nicotinic hydroxamate 11 d displayed sub-nanomolar potency (IC50 : 0.5 nM) and selectivity up to 34 000 times that of HDAC4 and from 100 to 1300 times that of all the other tested HDAC isoforms. The 2'-aminoanilides were class I-selective HDAC inhibitors, generally more potent against HDAC3, with the nicotinic anilide 12 d being the most effective (IC50 HDAC3 =0.113 μM). When tested in U937 leukemia cells, the hydroxamates 9 e, 11 c, and 11 d blocked over 80 % of cells in G2/M phase, whereas the anilides did not alter cell-cycle progress. In the same cell line, the hydroxamate 11 c and the anilide 10 b induced about 30 % apoptosis, and the anilide 12 c displayed about 40 % cytodifferentiation. Finally, the most potent compounds in leukemia cells 9 b, 11 c, 10 b, 10 e, and 12 c were also tested in K562, HCT116, and A549 cancer cells, displaying antiproliferative IC50 values at single-digit to sub-micromolar level.

In recent years, sirtuins (SIRTs) gained an increasing consideration because of their multiple key roles in several biological settings such as the regulation of transcription, energetic metabolism, cell cycle progression, and cytodifferentiation, apoptosis, neuro- and cardio-protection, inflammation, cancer onset and progression. Since there is mounting evidence in favor of potential therapeutic applications of SIRT modulators in various age-related disorders, the search about them is quite active. Areas covered: This review includes the patents regarding SIRT modulators released from 2015 to 2019 and provides an overview of the most relevant SIRT modulators.Expert opinion: Despite the knowledge about this family of broad-spectrum protein lysine deacylases has recently massively increased, there are still open questions, first of all, the exact nature of their involvement in various age-related conditions. The search for isoform-specific SIRT activators and inhibitors is still at its infancy, a limited number of patents describing them has been released, and not many clinical trials are ongoing. However, it is extremely likely that the successes obtained in the structural elucidation and structure-based design approaches that very recently have led to potent and specific SIRT modulators will pave the way for the development of further compounds selective for every single isoform.

Arylsulfatase A (ARSA) plays a crucial role in the reproduction of mammals due to its involvement in the specific gamete interaction preceding sperm and egg fusion leading to fertilization. Recently, it has been shown that zona pellucida (ZP) sperm binding and in vivo fertilization in mice are markedly hampered by using a specific anti-ARSA antibody. Herein, the design and discovery of the first ARSA small molecule inhibitor based on a coumarin-containing polycyde are presented. Through a structure-based approach applied on our in-house library, compound 1r was identified as an ARSA reversible inhibitor (ARSAi); then its activity was validated through both surface plasmon resonance and biochemical inhibition experiments, the first providing a K-D value of 21 mu M and the latter an IC50 value of 13.2 mu M. Further investigations highlighted that compound 1r induced 20% sperm death at 25 mu M and also impaired sperm motility; nevertheless both the effects were mediated by ROS production, since they were rescued by the cotreatment of 1r and N-acetyl cysteine (NAC). Interestingly, while 1r was not able to hamper the ZP/sperm binding, it markedly decreased the in vitro oocyte fertilization by mouse sperm up to 60%. Notably, this effect was not hampered by 1r/NAC coadministration, hence allowing the ruling out of an ROS-dependent mechanism. In conclusion, herein is reported the first ever hit of ARSAi as a chemical tool that will enable better exploration of ARSA's biological role in fertilization as well as provide a starting point for developing 1r structure optimization aimed at increasing enzyme inhibition potency but also providing a deeper understanding of the involvement of ARSA in the fertilization pathway mechanism.

[Image: see text] Since the histone modifying enzymes EZH2 and HDACs control a number of epigenetic-dependent carcinogenic pathways, we designed the first-in-class dual EZH2/HDAC inhibitor 5 displaying (sub)micromolar inhibition against both targets. When tested in several cancer cell lines, the hybrid 5 impaired cell viability at low micromolar level and in leukemia U937 and rhabdomyosarcoma RH4 cells provided G1 arrest, apoptotic induction, and increased differentiation, associated with an increase of acetyl-H3 and acetyl-α-tubulin and a decrease of H3K27me3 levels. In glioblastoma U87 cells, 5 hampered epithelial to mesenchymal transition by increasing the E-cadherin expression, thus proposing itself as a useful candidate for anticancer therapy.

DNA methyltransferases (DNMTs) play a relevant role in epigenetic control of cancer cell survival and proliferation. Since only two DNMT inhibitors (azacitidine and decitabine) have been approved to date for the treatment of hematological malignancies, the development of novel potent and specific inhibitors is urgent. Here we describe the design, synthesis, and biological evaluation of a new series of compounds acting at the same time as DNMTs (mainly DNMT3A) inhibitors and degraders. Tested against leukemic and solid cancer cell lines, 2a&ndash, c and 4a&ndash, c (the last only for leukemias) displayed up to submicromolar antiproliferative activities. In HCT116 cells, such compounds induced EGFP gene expression in a promoter demethylation assay, confirming their demethylating activity in cells. In the same cell line, 2b and 4c chosen as representative samples induced DNMT1 and -3A protein degradation, suggesting for these compounds a double mechanism of DNMT3A inhibition and DNMT protein degradation.

Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the Polycomb repressive complex 2 (PRC2), catalyzes the methylation of lysine 27 of histone H3 (H3K27) up to its trimethylated form (H3K27me), inducing by this way block of transcription and gene silencing. High levels of H3K27me3 have been found in both hematological malignancies and solid cancers, due to EZH2 overexpression and/or EZH2 mutation. From 2012, a number of highly potent and selective catalytic inhibitors of EZH2 have been reported, almost all bearing a 2-pyridone group in their structure. Typically, 2-pyridone inhibitors are selective for EZH2 over other methyltransferases, and some of them are specific for EZH2 over EZH1, others behave as dual EZH2/EZH1 inhibitors. The 2-pyridone moiety was crucial for the enzyme inhibition, as revealed later by crystallographic studies because it occupies partially the site for the co-substrate SAM (or the by-product, SAH) in the binding pocket of the enzyme, accounting for the SAM-competitive mechanism of action displayed by all the 2-pyridone inhibitors. The 2-pyridone warhead is linked to a support substructure, that can be either a bicyclic heteroaromatic ring (such as indazole, see for instance EPZ005687 and UNC1999, or indole, see for instance GSK126, EI1, and the more recent CPI-1205) or a simple monocyclic (hetero) aromatic ring (tazemetostat, MC3629, (R)-OR-S1/2), eventually annulated with the amide chain carrying the 2-pyridone group (3,4-dihydroisoquinoline-1(2H)-ones). Different substitutions at the support moiety influence the pharmacokinetics and pharmacodynamics of the compounds as well as their water solubility. In cancer diseases, the first reported 2-pyridone inhibitors displayed high antiproliferative effects in vitro and in vivo in lymphomas characterized by mutant EZH2 (such as Y641N), but the most recent compounds exert their anticancer activity against tumors with wild-type EZH2 as well. The dual EZH2/1 inhibitors have been recently reported to be more effective than EZH2 selective inhibitors in specific leukemias including leukemias cancer stem cells.

Antidepressant action has been linked to increased synaptic plasticity in which epigenetic mechanisms such as histone posttranslational acetylation could be involved. Interestingly, the histone deacetylases HDAC5 and SIRT2 are oppositely regulated by stress and antidepressants in mice prefrontal cortex (PFC). Besides, the neuroblastoma SH-SY5Y line is an in vitro neuronal model reliable to study drug effects with clear advantages over animals. We aimed to characterize in vitro the role of HDAC5 and SIRT2 in antidepressant regulation of neuroplasticity. SH-SY5Y cultures were incubated with imipramine, fluoxetine, and reboxetine (10 μM, 2 and 24 h) as well as the selective HDAC5 (MC3822, 5 μM, 24 h) or SIRT2 (33i, 5 μM, 24 h) inhibitors. The regulation of the brain-derived neurotrophic factor (BDNF), the vesicular glutamate transporter 1 (VGLUT1), the acetylated histones 3 (AcH3) and 4 (AcH4), HDAC5, and SIRT2 was studied. Comparatively, the long-term effects of these antidepressants (21 days, i.p.) in the mice (C57BL6, 8 weeks) PFC were studied. Antidepressants increased both in vitro and in vivo expression of BDNF, VGLUT1, AcH3, and AcH4. Moreover, imipramine and reboxetine increased the phosphorylated form of HDAC5 (P-HDAC5), mediating its cytoplasmic export. Further, SIRT2 was downregulated by all antidepressants. Finally, specific inhibition of HDAC5 and SIRT2 increased neuroplasticity markers. This study supports the validity of the SH-SY5Y model for studying epigenetic changes linked to synaptic plasticity induced by antidepressants as well as the effect of selective HDAC inhibitors. Particularly, nucleocytoplasmic export of HDAC5 and SIRT2 downregulation mediated by antidepressants could enhance synaptic plasticity markers leading to antidepressant action.

The implications of lysine-specific demethylase-1 (LSD1) in tumorigenesis have urged scientists to develop diagnostic tools in order to explore the function of this enzyme. In this work, we present our efforts on the development of tranylcypromine (TCP)-based functionalized probes for activity-based protein profiling (ABPP) of LSD1 activity. Biotinylated forms of selected compounds enabled dose-dependent enzyme labeling of recombinant LSD1. However, treatment with LSD1 inhibitors did not clearly reduce the LSD1 labeling efficiency thus indicating that labeling using these probes is not activity dependent. This calls for alternative strategies to develop probes for ABPP of the enzyme LSD1.

To identify the metabolite distribution in ascidian, we have applied an integrated liquid chromatography– tandem mass spectrometry (LC–MS) metabolomics approach to explore and identify patterns in chemical diversity of invasive ascidian Styela plicata. A total of 71 metabolites were reported among these alkaloids, fatty acids and lipids are the most dominant chemical group. Multivariate statistical analysis, principal component analysis (PCA) showed a clear separation according to chemical diversity and taxonomic groups. PCA and partial least square discriminant analysis were applied to discriminate the chemical group of S. plicata crude compounds and classify the compounds with unknown biological activities. In this study, we reported for the first time that a partially purified methanol extract prepared from the ascidian S. plicata and Ascidia mentula possess antitumor activity against four tumor cell lines with different tumor histotype, such as HeLa (cervical carcinoma), HT29 (colon carcinoma), MCF-7 (breast carcinoma) and M14 (melanoma). S. plicata fraction SP-50 showed strong inhibition of cell proliferation and induced apoptosis in HeLa and HT29 cells, thus indicating S. plicata fraction SP-50 a potential lead compound for anticancer therapy. The molecular mechanism of action and chemotherapeutic potential of these ascidian unknown biomolecules need further research.