Heterocycles-Containing HDAC Inhibitors Active in Cancer: An Overview of the Last Fifteen Years.

Cancer is one of the primary causes of mortality worldwide. Despite nowadays are numerous therapeutic treatments to fight tumor progression, it is still challenging to completely overcome it. It is known that Histone Deacetylases (HDACs), epigenetic enzymes that remove acetyl groups from lysines on histone's tails, are overexpressed in various types of cancer, and their inhibition represents a valid therapeutic strategy. To date, some HDAC inhibitors have achieved FDA approval. Nevertheless, several other potential drug candidates have been developed. This review aims primarily to be comprehensive of the studies done so far regarding HDAC inhibitors bearing heterocyclic rings since their therapeutic potential is well known and has gained increasing interest in recent years. Hence, inserting heterocyclic moieties in the HDAC-inhibiting scaffold can be a valuable strategy to provide potent and/or selective compounds. Here, in addition to summarizing the properties of novel heterocyclic HDAC inhibiting compounds, we also provide ideas for developing new, more potent, and selective compounds for treating cancer.

Correction: Zwergel et al. Novel Quinoline Compounds Active in Cancer Cells through Coupled DNA Methyltransferase Inhibition and Degradation. Cancers 2020, 12, 447.

In the original publication [...].

Mechanisms of mesothelial cell response to viral infections: HDAC1-3 inhibition blocks poly(I:C)-induced type I interferon response and modulates the mesenchymal/inflammatory phenotype.

Infectious peritonitis is a leading cause of peritoneal functional impairment and a primary factor for therapy discontinuation in peritoneal dialysis (PD) patients. Although bacterial infections are a common cause of peritonitis episodes, emerging evidence suggests a role for viral pathogens. Toll-like receptors (TLRs) specifically recognize conserved pathogen-associated molecular patterns (PAMPs) from bacteria, viruses, and fungi, thereby orchestrating the ensuing inflammatory/immune responses. Among TLRs, TLR3 recognizes viral dsRNA and triggers antiviral response cascades upon activation. Epigenetic regulation, mediated by histone deacetylase (HDAC), has been demonstrated to control several cellular functions in response to various extracellular stimuli. Employing epigenetic target modulators, such as epidrugs, is a current therapeutic option in several cancers and holds promise in treating viral diseases. This study aims to elucidate the impact of TLR3 stimulation on the plasticity of human mesothelial cells (MCs) in PD patients and to investigate the effects of HDAC1-3 inhibition. Treatment of MCs from PD patients with the TLR3 agonist polyinosinic:polycytidylic acid (Poly(I:C)), led to the acquisition of a bona fide mesothelial-to-mesenchymal transition (MMT) characterized by the upregulation of mesenchymal genes and loss of epithelial-like features. Moreover, Poly(I:C) modulated the expression of several inflammatory cytokines and chemokines. A quantitative proteomic analysis of MCs treated with MS-275, an HDAC1-3 inhibitor, unveiled altered expression of several proteins, including inflammatory cytokines/chemokines and interferon-stimulated genes (ISGs). Treatment with MS-275 facilitated MMT reversal and inhibited the interferon signature, which was associated with reduced STAT1 phosphorylation. However, the modulation of inflammatory cytokine/chemokine production was not univocal, as IL-6 and CXCL8 were augmented while TNF-α and CXCL10 were decreased. Collectively, our findings underline the significance of viral infections in acquiring a mesenchymal-like phenotype by MCs and the potential consequences of virus-associated peritonitis episodes for PD patients. The observed promotion of MMT reversal and interferon response inhibition by an HDAC1-3 inhibitor, albeit without a general impact on inflammatory cytokine production, has translational implications deserving further analysis.

SIRT3 Activation a Promise in Drug Development? New Insights into SIRT3 Biology and Its Implications on the Drug Discovery Process.

Sirtuins catalyze deacetylation of lysine residues with a NAD+-dependent mechanism. In mammals, the sirtuin family is composed of seven members, divided into four subclasses that differ in substrate specificity, subcellular localization, regulation, as well as interactions with other proteins, both within and outside the epigenetic field. Recently, much interest has been growing in SIRT3, which is mainly involved in regulating mitochondrial metabolism. Moreover, SIRT3 seems to be protective in diseases such as age-related, neurodegenerative, liver, kidney, heart, and metabolic ones, as well as in cancer. In most cases, activating SIRT3 could be a promising strategy to tackle these health problems. Here, we summarize the main biological functions, substrates, and interactors of SIRT3, as well as several molecules reported in the literature that are able to modulate SIRT3 activity. Among the activators, some derive from natural products, others from library screening, and others from the classical medicinal chemistry approach.

HDAC1/2 control mesothelium/ovarian cancer adhesive interactions impacting on Talin-1-α5ß1-integrin-mediated actin cytoskeleton and extracellular matrix protein remodeling.

BACKGROUND: Peritoneal metastasis, which accounts for 85% of all epithelial ovarian carcinoma (EOC) metastases, is a multistep process that requires the establishment of adhesive interactions between cancer cells and the peritoneal membrane. Interrelations between EOC and the mesothelial stroma are critical to facilitate the metastatic process. No data is available so far on the impact of histone acetylation/deacetylation, a potentially relevant mechanism governing EOC metastasis, on mesothelial cells (MCs)-mediated adhesion. METHODS: Static adhesion and peritoneal clearance experiments were performed pretreating mesenchymal-like MCs and platinum-sensitive/resistant EOC cell lines with MS-275-a Histone deacetylase (HDAC)1-3 pharmacological inhibitor currently used in combination trials. Results were acquired by confocal microscopy and were analyzed with an automated Opera software. The role of HDAC1/2 was validated by genetic silencing. The role of α4-, α5-α1 Integrins and Fibronectin-1 was validated using specific monoclonal antibodies. Quantitative proteomic analysis was performed on primary MCs pretreated with MS-275. Decellularized matrices were generated from either MS-275-exposed or untreated cells to study Fibronectin-1 extracellular secretion. The effect of MS-275 on ß1 integrin activity was assessed using specific monoclonal antibodies. The role of Talin-1 in MCs/EOC adhesion was analyzed by genetic silencing. Talin-1 ectopic expression was validated as a rescue tool from MS-275-induced phenotype. The in vivo effect of MS-275-induced MC remodeling was validated in a mouse model of peritoneal EOC dissemination. RESULTS: Treatment of MCs with non-cytotoxic concentrations of MS-275 caused a consistent reduction of EOC adhesion. Proteomic analysis revealed several pathways altered upon MC treatment with MS-275, including ECM deposition/remodeling, adhesion receptors and actin cytoskeleton regulators. HDAC1/2 inhibition hampered actin cytoskeleton polymerization by downregulating actin regulators including Talin-1, impairing ß1 integrin activation, and leading to abnormal extracellular secretion and distribution of Fibronectin-1. Talin-1 ectopic expression rescued EOC adhesion to MS-275-treated MCs. In an experimental mouse model of metastatic EOC, MS-275 limited tumor invasion, Fibronectin-1 secretion and the sub-mesothelial accumulation of MC-derived carcinoma-associated fibroblasts. CONCLUSION: Our study unveils a direct impact of HDAC-1/2 in the regulation of MC/EOC adhesion and highlights the regulation of MC plasticity by epigenetic inhibition as a potential target for therapeutic intervention in EOC peritoneal metastasis.

Histone deacetylase 10: A polyamine deacetylase from the crystal structure to the first inhibitors.

Polyamine deacetylase activity was discovered more than 40 years ago, but the responsible histone deacetylase 10 (HDAC10) was described only recently. HDAC10 is a class IIb HDAC, as is its closest relative, the α-tubulin deacetylase HDAC6. HDAC10 has attracted attention over the last 2 years due to its role in diseases, especially cancer. This review summarises chemical and structural biology approaches to the study of HDAC10. Light will be shed on recent advances in understanding the complex structural biology of HDAC10 and the discovery of the first highly selective HDAC10 inhibitors.

Novel 1,4-Dihydropyridines as Specific Binders and Activators of SIRT3 Impair Cell Viability and Clonogenicity and Downregulate Hypoxia-Induced Targets in Cancer Cells.

The mitochondrial SIRT3 modulates several biological pathways such as cancer, metabolism, and hypoxia-related diseases. Recently, we discovered new 1,4-dihydropyridines, compounds 2 and 3, the latter being a SIRT3-specific activator. In the present work, a novel 2- and 3-related small series of compounds have been developed, with 3c displaying the strongest SIRT3 binding and activation, with a KD of 29 µM and 387% of enzyme activation. Differently, 3d was the best in enhancing glutamate dehydrogenase activity and deacetylating K68- and K122-acMnSOD in triple-negative MDA-MB-231 breast cancer cells. Tested in CAL-62 thyroid cancer and MDA-MB-231 cells, 3d displayed the strongest time- and dose-dependent reduction of cell viability and clonogenicity at a single-digit micromolar level, along with cell death, in both normoxia and hypoxia conditions. Moreover, 3d downregulated not only hypoxia-induced factors, such as HIF-1α, EPAS-1, and CA-IX, but also epithelial-mesenchymal transition master regulators and extracellular matrix components such as SNAIL1, ZEB1, SLUG, COL1A2, MMP2, and MMP9, markedly hampering MDA-MB-231 cell migration.

Novel pyridine-containing histone deacetylase inhibitors strongly arrest proliferation, induce apoptosis and modulate miRNAs in cancer cells.

After over 30 years of research, the development of HDAC inhibitors led to five FDA/Chinese FDA-approved drugs and many others under clinical or preclinical investigation to treat cancer and non-cancer diseases. Herein, based on our recent development of pyridine-based isomers as HDAC inhibitors, we report a series of novel 5-acylamino-2-pyridylacrylic- and -picolinic hydroxamates and 2'-aminoanilides 5-8 as anticancer agents. The hydroxamate 5d proved to be quite HDAC3/6-selective exhibiting IC50 values of 80 and 11 nM, respectively, whereas the congener 5e behaved as inhibitor of HDAC1-3, -6, -8, and -10 (class I/IIb-selective inhibitor) at nanomolar level. Compound 5e provided a huge antiproliferative activity (nanomolar IC50 values) against both haematological and solid cancer cell lines. In leukaemia U937 cells, the hydroxamate 5d and the 2'-aminoanilide 8f induced remarkable cell death after 48 h, with 76% and 100% pre-G1 phase arrest, respectively, showing a stronger effect with respect to SAHA and MS-275 used as reference compounds. In U937 cells, the highest dose- and time-dependent cytodifferentiation was obtained by the 2'-aminoanilide 8d (up to 35% of CD11c positive/propidium iodide negative cells at 5 µM for 48 h). The same 8d and the hydroxamates 5d and 5e were the most effective in inducing p21 protein expression in the same cell line. Mechanistically, 5d, 5e, 8d and 8f increased mRNA expression of p21, BAX and BAK, downregulated cyclin D1 and BCL-2 and modulated pro- and anti-apoptotic microRNAs towards apoptosis induction. Finally, 5e strongly arrested proliferation in nine different haematological cancer cell lines, with dual-digit nanomolar potency towards MV4-11, Kasumi-1, and NB4, being more potent than mocetinostat, used as reference drug.

PD-L1 small-molecule modulators: A new hope in epigenetic-based multidrug cancer therapy?

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.

HDAC1-3 inhibition increases SARS-CoV-2 replication and productive infection in lung mesothelial and epithelial cells.

Background: Despite the significant progress achieved in understanding the pathology and clinical management of SARS-CoV-2 infection, still pathogenic and clinical issues need to be clarified. Treatment with modulators of epigenetic targets, i.e., epidrugs, is a current therapeutic option in several cancers and could represent an approach in the therapy of viral diseases. Results: Aim of this study was the analysis of the role of histone deacetylase (HDAC) inhibition in the modulation of SARS-CoV-2 infection of mesothelial cells (MCs).MeT5A cells, a pleura MC line, were pre-treated with different specific class I and IIb HDAC inhibitors. Unexpectedly, treatment with HDAC1-3 inhibitors significantly increased ACE2/TMPRSS2 expression, suggesting a role in favoring SARS-CoV-2 infection. We focused our analysis on the most potent ACE2/TMPRSS2 inducer among the inhibitors analysed, MS-275, a HDAC1-3 inhibitor. ACE2/TMPRSS2 expression was validated by Western Blot (WB) and immunofluorescence. The involvement of HDAC inhibition in receptor induction was confirmed by HDAC1/HDAC2 silencing. In accordance to the ACE2/TMPRSS2 expression data, MS-275 increased SARS-CoV-2 replication and virus propagation in Vero E6 cells.Notably, MS-275 was able to increase ACE2/TMPRSS2 expression and SARS-CoV-2 production, although to a lesser extent, also in the lung adenocarcinoma cell line Calu-3 cells.Mechanistically, treatment with MS-275 increased H3 and H4 histone acetylation at ACE2/TMPRSS2 promoters, increasing their transcription. Conclusion: This study highlights a previously unrecognized effect of HDAC1-3 inhibition in increasing SARS-CoV-2 cell entry, replication and productive infection correlating with increased expression of ACE2 and TMPRSS2. These data, while adding basic insight into COVID-19 pathogenesis, warn for the use of HDAC inhibitors in SARS-CoV-2 patients.

Restoration of WT1/miR-769-5p axis by HDAC1 inhibition promotes MMT reversal in mesenchymal-like mesothelial cells.

Histone acetylation/deacetylation play an essential role in modifying chromatin structure and in regulating cell plasticity in eukaryotic cells. Therefore, histone deacetylase (HDAC) pharmacological inhibitors are promising tools in the therapy of fibrotic diseases and in cancer. Peritoneal fibrosis is a pathological process characterized by many cellular and molecular alterations, including the acquisition of invasive/pro-fibrotic abilities by mesothelial cells (MCs) through induction of mesothelial to mesenchymal transition (MMT). The aim of this study was to characterize the molecular mechanism of the antifibrotic role of HDAC1 inhibition. Specifically, treatment with MS-275, an HDAC1-3 inhibitor previously known to promote MMT reversal, induced the expression of several TGFBRI mRNA-targeting miRNAs. Among them, miR-769-5p ectopic expression was sufficient to promote MMT reversal and to limit MC migration and invasion, whereas miR-769-5p silencing further enhanced mesenchymal gene expression. These results were confirmed by HDAC1 genetic silencing. Interestingly, miR-769-5p silencing maintained mesenchymal features despite HDAC1 inhibition, thus indicating that it is necessary to drive MMT reversal induced by HDAC1 inhibition. Besides TGFBRI, miR-769-5p was demonstrated to target SMAD2/3 and PAI-1 expression directly. When analyzing molecular mechanisms underlying miR-769-5p expression, we found that the transcription factor Wilms' tumor 1 (WT1), a master gene controlling MC development, binds to the miR-769-5p promoter favoring its expression. Interestingly, both WT1 expression and binding to miR-769-5p promoter were increased by HDAC1 inhibition and attenuated by TGFß1 treatment. Finally, we explored the significance of these observations in the cell-to-cell communication: we evaluated the ability of miR-769-5p to be loaded into extracellular vesicles (EVs) and to promote MMT reversal in recipient mesenchymal-like MCs. Treatment of fibrotic MCs with EVs isolated from miR-769-5p over-expressing MCs promoted the down-regulation of specific mesenchymal targets and the reacquisition of an epithelial-like morphology. In conclusion, we highlighted an HDAC1-WT1-miR-769-5p axis potentially relevant for therapies aimed at counteracting organ fibrosis.

Potent and Specific Activators for Mitochondrial Sirtuins Sirt3 and Sirt5.

Sirtuins are NAD+-dependent protein deacylases involved in metabolic regulation and aging-related diseases. Specific activators for seven human Sirtuin isoforms would be important chemical tools and potential therapeutic drugs. Activators have been described for Sirt1 and act via a unique N-terminal domain of this isoform. For most other Sirtuin isoforms, including mitochondrial Sirt3-5, no potent and specific activators have yet been identified. We here describe the identification and characterization of 1,4-dihydropyridine-based compounds that either act as pan Sirtuin activators or specifically stimulate Sirt3 or Sirt5. The activators bind to the Sirtuin catalytic cores independent of NAD+ and acylated peptides and stimulate turnover of peptide and protein substrates. The compounds also activate Sirt3 or Sirt5 in cellular systems regulating, e.g., apoptosis and electron transport chain. Our results provide a scaffold for potent Sirtuin activation and derivatives specific for Sirt3 and Sirt5 as an excellent basis for further drug development.

Seleno-vs. thioether triazine derivatives in search for new anticancer agents overcoming multidrug resistance in lymphoma.

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 (IC50: 16.73 µM) as well as concerning the antiproliferative effect (IC50: 5.35 µM) in MDR cells. Regarding the mechanistic studies looking at the cell cycle, the thioether 15 and selenium derivatives 26 and 29 were significantly effective in the regulation of cell cycle-related genes alone or in co-treatment with doxorubicin counteracting Cyclin D1 and E1 expression and increasing p53 and p21 levels, shedding first light on their mechanism of action. In summary, we explored the chemical space of seleno- and thioether 1,3,5-triazine derivatives with interesting activity against lymphoma. Especially compound 15 is worthy of being studied deeper to evaluate its precise mode of action further as well it can be improved regarding its potency and drug-likeness.

Heterocycle-containing tranylcypromine derivatives endowed with high anti-LSD1 activity.

As regioisomers/bioisosteres of 1a, a 4-phenylbenzamide tranylcypromine (TCP) derivative previously disclosed by us, we report here the synthesis and biological evaluation of some (hetero)arylbenzoylamino TCP derivatives 1b-6, in which the 4-phenyl moiety of 1a was shifted at the benzamide C3 position or replaced by 2- or 3-furyl, 2- or 3-thienyl, or 4-pyridyl group, all at the benzamide C4 or C3 position. In anti-LSD1-CoREST assay, all the meta derivatives were more effective than the para analogues, with the meta thienyl analogs 4b and 5b being the most potent (IC50 values = 0.015 and 0.005 µM) and the most selective over MAO-B (selectivity indexes: 24.4 and 164). When tested in U937 AML and prostate cancer LNCaP cells, selected compounds 1a,b, 2b, 3b, 4b, and 5a,b displayed cell growth arrest mainly in LNCaP cells. Western blot analyses showed increased levels of H3K4me2 and/or H3K9me2 confirming the involvement of LSD1 inhibition in these assays.

Selenium-Containing Agents Acting on Cancer-A New Hope?

Selenium-containing agents are more and more considered as an innovative potential treatment option for cancer. Light is shed not only on the considerable advancements made in understanding the complex biology and chemistry related to selenium-containing small molecules but also on Se-nanoparticles. Numerous Se-containing agents have been widely investigated in recent years in cancer therapy in relation to tumour development and dissemination, drug delivery, multidrug resistance (MDR) and immune system-related (anti)cancer effects. Despite numerous efforts, Se-agents apart from selenocysteine and selenomethionine have not yet reached clinical trials for cancer therapy. The purpose of this review is to provide a concise critical overview of the current state of the art in the development of highly potent target-specific Se-containing agents.

Polycomb Repressive Complex 2 Modulation through the Development of EZH2-EED Interaction Inhibitors and EED Binders.

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 RNA methylation and beyond - The epigenetic machinery and potential treatment options.

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.

Structure-Guided Development of Small-Molecule PRC2 Inhibitors Targeting EZH2-EED Interaction.

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 Å. 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.