Anticancer effects of the HDAC inhibitor, 3ß,6ß­dihydroxyurs­12­en­27­oic acid, in MCF­7 breast cancer cells via the inhibition of Akt/mTOR pathways.

Astilbe chinensis (A. chinensis) is a perennial herb that is used to treat chronic bronchitis and pain. The anticancer activity of 3ß,6ß­dihydroxyurs­12­en­27­oic acid (ACT­3), a major component isolated from A. chinensis, has not yet been investigated in detail. The purpose of the present study was to investigate the histone deacetylase (HDAC) inhibitory and anticancer activities of ACT­3 compared with suberoylanilide hydroxamic acid (SAHA) in MCF­7 human breast cancer cells. The purity of ACT­3 was determined using high­performance liquid chromatography. In the present study, the effects of ACT­3 on anticancer effects of MCF­7 cells were determined by measuring the level of apoptotic cell death and cell cycle regulator using flow cytometry analysis and western blot analysis, respectively. The effects of ACT­3 on HDAC enzyme activity were measured using assay kits. ACT­3 and SAHA increased the levels of acetylated histone H3 and reduced the levels of HDAC1 and HDAC3 in MCF­7 cells. ACT­3 significantly decreased the cell viability in a concentration­dependent manner and induced different morphological changes at high concentrations. ACT­3 and SAHA significantly inhibited the colony formation in MCF­7 cells. ACT­3 inhibited total HDAC activity in a dose­dependent manner. ACT­3 significantly reduced the expression levels of cyclin D1 and cyclin­dependent kinase 4, and upregulated the expression levels of p21WAF1 and p53. A significant increase in the G1 phase cell population was observed in MCF­7 cells and ACT­3 induced apoptosis by reducing the ratio of B­cell lymphoma­2 (Bcl­2)/Bcl­2­associated X (Bax) and releasing cleaved caspase 9. Additionally, ACT­3 significantly increased autophagic cell death by inhibiting the serine­threonine kinase/mammalian target of the rapamycin pathway. Autophagy induction was confirmed via acridine orange staining. ACT­3 significantly increased the pERK1/2 and p21 in MCF­7 cells. Thus, the activated ERK pathway played an important role in cell cycle arrest and apoptosis via ERK­dependent induction of p21 in MCF­7 cells. These data indicated that ACT­3 can be used as a promising anticancer agent to overcome the limitations and reduce the side effects of conventional anticancer drugs.

Ulleunganilines A-C, Trichostatin Analogues Bearing a Modified Side Chain from Streptomyces sp. 13F051.

Three new trichostatin analogues, ulleunganilines A-C (1-3), and seven known trichostatins (4-10) were isolated from cultures of Streptomyces sp. 13F051. NMR, UV, and MS data indicated that the planar structures of 1-3 consisted of modified side chains in the trichostatic acid moiety. The absolute configuration of the 2,4-dimethyl-branched carbon chains in 1 and 2 was determined by the PGME method, while the amino acid group in 3 was identified by advanced Marfey's method. Based on the structure of the modified side chains, the origin of 1-3 is proposed. Further experiments indicated that 1 and 3 displayed moderate histone deacetylase inhibitory activity, suggesting that not only the hydroxamate group but also the N,N-dimethyl group were essential for the inhibitory activity.

Identification of potential chemical compounds enhancing generation of enucleated cells from immortalized human erythroid cell lines.

Immortalized erythroid cell lines are expected to be a promising source of ex vivo manufactured red blood cells (RBCs), however the induction of enucleation in these cell lines is inefficient at present. We utilized an imaging-based high-throughput system to identify chemical compounds that trigger enucleation of human erythroid cell lines. Among >3,300 compounds, we identified multiple histone deacetylase inhibitors (HDACi) inducing enucleated cells from the cell line, although an increase in membrane fragility of enucleated cells was observed. Gene expression profiling revealed that HDACi treatment increased the expression of cytoskeletal genes, while an erythroid-specific cell membrane protein, SPTA1, was significantly down-regulated. Restoration of SPTA1 expression using CRISPR-activation partially rescued the fragility of cells and thereby improved the enucleation efficiency. Our observations provide a potential solution for the generation of mature cells from erythroid cell lines, contributing to the future realization of the use of immortalized cell lines for transfusion therapies.

Natural Products Extracted from Fungal Species as New Potential Anti-Cancer Drugs: A Structure-Based Drug Repurposing Approach Targeting HDAC7.

Mushrooms can be considered a valuable source of natural bioactive compounds with potential polypharmacological effects due to their proven antimicrobial, antiviral, antitumor, and antioxidant activities. In order to identify new potential anticancer compounds, an in-house chemical database of molecules extracted from both edible and non-edible fungal species was employed in a virtual screening against the isoform 7 of the Histone deacetylase (HDAC). This target is known to be implicated in different cancer processes, and in particular in both breast and ovarian tumors. In this work, we proposed the ibotenic acid as lead compound for the development of novel HDAC7 inhibitors, due to its antiproliferative activity in human breast cancer cells (MCF-7). These promising results represent the starting point for the discovery and the optimization of new HDAC7 inhibitors and highlight the interesting opportunity to apply the "drug repositioning" paradigm also to natural compounds deriving from mushrooms.

Epigallocatechin-3-gallate (EGCG) Alters Histone Acetylation and Methylation and Impacts Chromatin Architecture Profile in Human Endothelial Cells.

Epigallocatechin gallate (EGCG), the main green tea polyphenol, exerts a wide variety of biological actions. Epigenetically, the catechin has been classified as a DNMTs inhibitor, however, its impact on histone modifications and chromatin structure is still poorly understood. The purpose of this study was to find the impact of EGCG on the histone posttranslational modifications machinery and chromatin remodeling in human endothelial cells of both microvascular (HMEC-1) and vein (HUVECs) origin. We analyzed the methylation and acetylation status of histones (Western blotting), as well as assessed the activity (fluorometric assay kit) and gene expression (qPCR) of the enzymes playing a prominent role in shaping the human epigenome. The performed analyses showed that EGCG increases histone acetylation (H3K9/14ac, H3ac), and methylation of both active (H3K4me3) and repressive (H3K9me3) chromatin marks. We also found that the catechin acts as an HDAC inhibitor in cellular and cell-free models. Additionally, we observed that EGCG affects chromatin architecture by reducing the expression of heterochromatin binding proteins: HP1α, HP1γ. Our results indicate that EGCG promotes chromatin relaxation in human endothelial cells and presents a broad epigenetic potential affecting expression and activity of epigenome modulators including HDAC5 and 7, p300, CREBP, LSD1 or KMT2A.

Screening and Phenotypical Characterization of Schistosoma mansoni Histone Deacetylase 8 (SmHDAC8) Inhibitors as Multistage Antischistosomal Agents.

Schistosomiasis (also known as bilharzia) is a neglected tropical disease caused by platyhelminths of the genus Schistosoma. The disease is endemic in tropical and subtropical areas of the world where water is infested by the intermediate parasite host, the snail. More than 800 million people live in endemic areas and more than 200 million are infected and require treatment. Praziquantel (PZQ) is the drug of choice for schistosomiasis treatment and transmission control being safe and very effective against adult worms of all the clinically relevant Schistosoma species. Unfortunately, it is ineffective on immature, juvenile worms; therefore, it does not prevent reinfection. Moreover, the risk of development and spread of drug resistance because of the widespread use of a single drug in such a large population represents a serious threat. Therefore, research aimed at identifying novel drugs to be used alone or in combination with PZQ are needed. Schistosoma mansoni histone deacetylase 8 (SmHDAC8) is a class I zinc-dependent HDAC, which is abundantly expressed in all stages of its life cycle, thus representing an interesting target for drug discovery. Through virtual screening and phenotypical characterization of selected hits, we discovered two main chemical classes of compounds characterized by the presence of a hydroxamate-based metal binding group coupled to a spiroindoline or a tricyclic thieno[3,2-b]indole core as capping groups. Some of the compounds of both classes were deeply investigated and showed to impair viability of larval, juvenile, and adult schistosomes, to impact egg production in vitro and/or to induce morphological alterations of the adult schistosome reproductive systems. Noteworthy, all of them inhibit the recombinant form of SmHDAC8 enzyme in vitro. Overall, we identified very interesting scaffolds, paving the way to the development of effective antischistosomal agents.

A Comparative Study of Target Engagement Assays for HDAC1 Inhibitor Profiling.

Histone deacetylases (HDACs) are epigenetic modulators linked to diseases including cancer and neurodegeneration. Given their therapeutic potential, highly sensitive biochemical and cell-based profiling technologies have been developed to discover small-molecule HDAC inhibitors. Ultimately, the therapeutic action of these inhibitors is dependent on a physical engagement with their intended targets in cellular and tissue environments. Confirming target engagement in the cellular environment is particularly relevant for HDACs since they function as part of cell type-specific multiprotein complexes. Here we implemented two recently developed high-throughput target engagement technologies, NanoBRET and SplitLuc CETSA, to profile 349 compounds in the Epigenetic-Focused collection for HDAC1 binding. We found that the two HDAC1 target engagement assays correlated well with each other and with orthogonal activity-based assays, in particular those carried out in cellular environments rather than with isolated HDAC proteins. The assays detected a majority of the previously described HDAC1 inhibitors in the collection and, importantly, triaged HDAC inhibitors known to target other HDACs.

The Development Process: from SAHA to Hydroxamate HDAC Inhibitors with Branched CAP Region and Linear Linker.

Histone deacetylases (HDACs) belong to a group of epigenetic regulatory enzymes that participate in modulating the acetylation level of histone lysine residues as well as non-histone proteins, and they play a key role in the regulation of gene expression. HDACs are potential anticancer drug targets highly expressed in various kinds of cancer cells. So far, five small molecules targeting HDACs have been approved for the therapy of cancer, and over 20 inhibitors of HDACs are under different phases of clinical trials. Among them, hydroxamate-based HDAC inhibitors (HDACis) represent a well-investigated series of chemical entities. The current review covers the recent progress in the discovery process, form SAHA to hydroxamate HDAC inhibitors with branched CAP region and linear linker. At the same time, the pharmacological and structure-activity relationship (SAR) studies of the specific derivatives from SAHA and the HDACis with branched CAP region and linear linker are also introduced.

A whole organism small molecule screen identifies novel regulators of pancreatic endocrine development.

An early step in pancreas development is marked by the expression of the transcription factor Pdx1 within the pancreatic endoderm, where it is required for the specification of all endocrine cell types. Subsequently, Pdx1 expression becomes restricted to the ß-cell lineage, where it plays a central role in ß-cell function. This pivotal role of Pdx1 at various stages of pancreas development makes it an attractive target to enhance pancreatic ß-cell differentiation and increase ß-cell function. In this study, we used a newly generated zebrafish reporter to screen over 8000 small molecules for modulators of pdx1 expression. We found four hit compounds and validated their efficacy at different stages of pancreas development. Notably, valproic acid treatment increased pancreatic endoderm formation, while inhibition of TGFß signaling led to α-cell to ß-cell transdifferentiation. HC toxin, another HDAC inhibitor, enhances ß-cell function in primary mouse and human islets. Thus, using a whole organism screening strategy, this study identified new pdx1 expression modulators that can be used to influence different steps in pancreas and ß-cell development.

Phytosterols and triterpenes from Morinda lucida Benth. exhibit binding tendency against class I HDAC and HDAC7 isoforms.

The important role of histone deacetylases (HDACs) in the development of cancer has been demonstrated by various studies. Thus targeting HDACs with inhibitors is a major focus in anticancer drug research. Although few synthetic HDAC inhibitors (HDIs) have been approved for cancer treatment, they have significant undesirable side effects. Therefore emphases have been placed on natural HDIs as substitutes for the synthetic ones. In a bid to identify more HDIs, this study evaluated the binding tendency of compounds derived from Morinda lucida Benth. towards selected HDACs for the discovery of potent HDIs as potential candidates for anticancer therapeutics, based on the report of anticancer potentials of Morinda lucida-derived extracts and compounds. Givinostat and 49 Morinda-lucida derived compounds were docked against selected HDAC isoforms using AutodockVina, while binding interactions were viewed with Discovery Studio Visualizer, BIOVIA, 2016. Druglikeness and Absorption-Distribution-Metabolism-Excretion (ADME) parameters of the top 7 compounds were evaluated using the Swiss online ADME web tool. The results revealed that out of the 49 compounds, 3 phytosterols (campesterol, cycloartenol, and stigmasterol) and 2 triterpenes (oleanolic acid and ursolic acid) exhibited high HDAC inhibitory activity compared to givinostat. These 5 compounds also fulfill oral drugability of Lipinski rule of five. Morinda lucida-derived phytosterols and triterpenes show high binding tendency towards the selected HDACs and exhibited good drugability characteristics and are therefore good candidates for further studies in the search for therapies against abnormalities linked with over-activity of HDACs.

Weighted gene co-expression network analysis and connectivity map identifies lovastatin as a treatment option of gastric cancer by inhibiting HDAC2.

OBJECTIVE: This study aimed to identifying and validating therapeutic compounds which might have positive effects on patients with gastric cancer (GC) based on weighted gene co-expression network analysis (WGCNA) and connectivity map (CMap). METHODS: We performed WGCNA to gain insights into the molecular aspects of GC. Raw microarray datasets (including 132 samples) were downloaded from the Gene Expression Omnibus (GEO) website. We utilized the WGCNA to identify the coexpressed genes (modules) and modular hub genes after non-specific filtering. Furthermore, these differentially expressed genes were submitted to CMap analysis to identify candidate therapeutic compounds for GC. In experimental part, cell growth inhibition was evaluated by Cell Counting Kit-8 (CCK-8) and colony formation assays. Tumor growth was assessed using nude mice with xenografts established in vivo. QRT-PCR and western blot were used for determination of HDAC2 expression level and immunohistochemistry was performed to quantify HDAC2 in gastric tumor samples. RESULTS: Through WGCNA and CMap analysis, we found two potential therapeutic compounds, the valproic acid (VPA), which is the histone deacetylase (HDAC) inhibitor and lovastatin. HDAC2 was overexpressed in gastric cancer cell lines including AGS, BGC-823, NCI-N87 and MKN28. Dose-dependent inhibition of gastric cancer cells by VPA and lovastatin was verified in vitro. Apoptosis of GC cells was induced after treatment with VPA and lovastatin through suppressing HDAC2 expression. Furthermore, the inhibition of VPA with cisplatin and lovastatin with cisplatin were also dose-dependent and cisplatin exhibited synergistic effects. In the xenografts, similar results were found. CONCLUSION: WGCNA was able to identify significant groups of genes associated with cancer prognosis. Moreover, analysis of gene expression signature using CMap is a powerful way to explore potential therapeutics for human diseases. For treating GC, lovastatin may be a potential drug.

Computational QSAR model combined molecular descriptors and fingerprints to predict HDAC1 inhibitors.

The dynamic balance between acetylation and deacetylation of histones plays a crucial role in the epigenetic regulation of gene expression. It is equilibrated by two families of enzymes: histone acetyltransferases and histone deacetylases (HDACs). HDACs repress transcription by regulating the conformation of the higher-order chromatin structure. HDAC inhibitors have recently become a class of chemical agents for potential treatment of the abnormal chromatin remodeling process involved in certain cancers. In this study, we constructed a large dataset to predict the activity value of HDAC1 inhibitors. Each compound was represented with seven fingerprints, and computational models were subsequently developed to predict HDAC1 inhibitors via five machine learning methods. These methods include naïve Bayes, κ-nearest neighbor, C4.5 decision tree, random forest, and support vector machine (SVM) algorithms. The best predicting model was CDK fingerprint with SVM, which exhibited an accuracy of 0.89. This model also performed best in five-fold cross-validation. Some representative substructure alerts responsible for HDAC1 inhibitors were identified by using MoSS in KNIME, which could facilitate the identification of HDAC1 inhibitors.

6- and 8-Prenylnaringenin, Novel Natural Histone Deacetylase Inhibitors Found in Hops, Exert Antitumor Activity on Melanoma Cells.

BACKGROUND/AIMS: Prenylnaringenins are natural prenylflavonoids with anticancer properties. However, the underlying mechanisms have not been elucidated yet. Here we report a novel mode of action of 6- and 8-prenylnaringenin (PN) on human melanoma cells: Inhibition of cellular histone deacetylases (HDACs). METHODS: We performed in silico and in vitro analyses using 6-PN or 8-PN to study a possible interaction of 6-PN or 8-PN with HDAC as well as Western blot and FACS analyses, real-time cell proliferation and cell viability assays to assess the impact of 6-PN and 8-PN on human metastatic melanoma cells. RESULTS: In silico, 6-PN and 8-PN fit into the binding pocket of HDAC2, 4, 7 and 8, binding to the zinc ion of their catalytic center that is essential for enzymatic activity. In vitro, 100 µmol/L of 6-PN or 8-PN inhibited all 11 conserved human HDAC of class I, II and IV. In clinical oncology HDAC inhibitors are currently investigated as new anticancer compounds. In line, treatment of SK-MEL-28 cells with 6-PN or 8-PN induced a hyperacetylation of histone complex H3 within 2 h. Further, 6-PN or 8-PN mediated a prominent, dose-dependent reduction of cellular proliferation and viability of SK-MEL-28 and BLM melanoma cells. This effect was apoptosis-independent and accompanied by down-regulation of mTOR-specific pS6 protein via pERK/pP90 in SK-MEL-28 cells. CONCLUSION: The identification of a broad inhibitory capacity of 6-PN and 8-PN for HDAC enzymes with antiproliferative effects on melanoma cells opens the perspective for clinical application as novel anti-melanoma drugs and the usage as innovative lead structures for chemical modification to enhance pharmacology or inhibitory activities.

Development of an Efficient Screening System for HDAC Inhibitor Based on TCF Response Element.

BACKGROUND: Acetylation of core histones is governed by opposing actions of a variety of histone acetyltransferases (HATs) and Histone Deacetylases (HDACs). Deregulation of histone acetylation has been causally linked to cancer development and progression, and can be potentially reversed by drug treatments. HDAC inhibitors (HDACis) have the anti-cancer effects such as induction of cell cycle arrest, promotion of cell apoptosis, and inhibition of metastasis. Many of these HDACis are currently in clinical development. METHODS: To screen for novel compound with HDACis activity, cell-based strategy has been developed in current work. The interaction of HDACs and TCFs was determined by reporter assay, and Natural products library (A10) was screened by our system in colon cancer cells. RESULTS: Based on the interaction of HDACs and TCFs, compounds with HDACis activity could be easily picked out from Natural products library (A10) by using this screening system. Finally, a compound Wsu-18 was preliminarily identified as a potential HDACis. CONCLUSION: These data suggest our screening system for HDACis was efficient and promising, and it is suitable for screening other small molecular chemical libraries.

Biochemical and Anti-Triple Negative Metastatic Breast Tumor Cell Properties of Psammaplins.

Breast tumors reprogram their cellular metabolism, nutrient uptake, and utilization-associated biochemical processes. These processes become further transformed as genetically predisposed metastatic breast tumor cells colonize specific organs. Breast tumor cells often metastasize to the brain, bone, lung and liver. Massagué and colleagues isolated organotropic subclones and established organ-specific gene signatures associated with lung-, bone-, and brain-specific metastatic triple-negative breast cancer (TNBC) MDA-MB-231 cells. Using these genetically characterized metastatic subclones specific to lung (LM4175), bone (BoM1833), and brain (BrM-2a), we evaluated marine natural products for the ability to differentially suppress metastatic breast cancer cells in a target organ-dependent manner. Psammaplin-based histone deacetylase (HDAC) inhibitors were found to differentially inhibit HDAC activity, induce activation of hypoxia-inducible factor-1 (HIF-1), and disrupt organotropic metastatic TNBC subclone growth. Further, psammaplins distinctly suppressed the outgrowth of BoM1833 tumor spheroids in 3D-culture systems. Similar results were observed with the prototypical HDAC inhibitor trichostatin A (TSA). These organotropic tumor cell-based studies suggest the potential application of HDAC inhibitors that may yield new directions for anti-metastatic breast tumor research and drug discovery.

Metabolites from the Dark Septate Endophyte Drechslera sp. Evaluation by LC/MS and Principal Component Analysis of Culture Extracts with Histone Deacetylase Inhibitors.

Secondary metabolites from the cultures of the dark septate fungal endophyte (DSE) Drechslera sp., isolated from the roots of rye grass (Lollium sp.) and cultured under different experimental conditions, are described here for the first time. The use of suberoylanilidehydroxamic acid (SAHA) and other histone deacetylase inhibitors as epigenetic modifiers in the culture medium was evaluated by LC/MS and LC/MS/MS. Several differences in the metabolite production were detected by means of supervised principal component analysis (PCA) of LC/MS data. The presence of the compounds in the culture medium or in the mycelium was compared. In order to confirm their structure, many of these natural products were isolated from a larger scale culture. These metabolites were characterized as prenylhydroxybenzoic acids and chromans, two compounds, one of each class were previously undescribed, prenylquinoids, diketopiperazines and macrosphelides. Some of the compounds, which were released to the medium, showed good antifungal activity, suggesting that these compounds could protect Lollium from fungal phytopatogens. The use of SAHA as an additive of the cultures also induced the release of hexosylphytosphyngosine to the culture medium. The biotransformation of the inhibitors was observed in addition to the production of antifungal metabolites, showing the ability of this endophytic strain to control xenobiotics.

Biophenols: Enzymes (ß-secretase, Cholinesterases, histone deacetylase and tyrosinase) inhibitors from olive (Olea europaea L.).

The focus of this study was on inhibition of enzymes involved in the pathogenesis Alzheimer's disease (AD) including prime amyloid beta (Aß) producing enzyme (ß-secretase: BACE-1) and disease progression enzymes including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), histone deacetylase (HDAC), and tyrosinase along with the catecholamine L-DOPA, by using olive biophenols. Here we report the strongest inhibition of BACE-1 from rutin (IC50: 3.8 nM) followed by verbascoside (IC50: 6.3 nM) and olive fruit extract (IC50: 18 ng), respectively. Olive biophenol, quercetin exhibited strongest enzyme inhibitory activity against tyrosinase (IC50: 10.73 µM), BChE (IC50: 19.08 µM), AChE (IC50: 55.44 µM), and HDAC (IC50: 105.1 µM) enzymes. Furthermore, olive biophenol verbascoside (IC50: 188.6 µM), and hydroxytyrosol extreme extract (IC50: 66.22 µg) were showed the highest levels of inhibition against the HDAC enzyme. Neuroprotective capacity against levodopa-induced toxicity in neuroblastoma (SH-SY5Y) cells of olive biophenols were assessed, where rutin indicated the highest neuroprotection (74%), followed by caffeic acid (73%), and extract hydroxytyrosol extreme (97%), respectively. To the best of our knowledge, this is the first in vitro report on the enzymes inhibitory activity of olive biophenols. Taken together, our in vitro results data suggest that olive biophenols could be a promising natural inhibitor, which may reduce the enzyme-induced toxicity associated with the oxidative stress involved in the progression of AD. CHEMICAL COMPOUNDS USED IN THE STUDY: Acetylthiocholine iodide (PubChem CID: 74629); S-Butyrylthiocholine chloride (PubChem CID: 3015121); Caffeic acid (PubChem CID: 689043); Dimethyl sulfoxide (DMSO) (PubChem: 679); L-3,4-Dihydroxyphenylalanine (L-DOPA) (PubChem CID: 6047); 5,5'-Dithiobis (2-nitrobenzoic acid) (DTNB) (PubChem CID: 6254); Epigallocatechin gallate (EGCG) (PubChem CID: 65064); Ethylenediamine tetraacetic acid (EDTA) (PubChem CID: 6049); Galantamine hydrobromide (PubChem CID: 121587); l-Glutamine (PubChem CID: 5961); Hydroxytyrosol (PubChem CID: 82755); Kojic acid (PubChem CID: 3840); Luteolin (PubChem CID: 5280445); Oleuropein (PubChem CID: 5281544); Penicillin-streptomycin (PubChem CID: 131715954); Quercetin (PubChem CID: 5280343); Rutin (PubChem CID: 5280805); Tris-HCl buffer (PubChem: 93573); Trypan blue (PubChem: 9562061).

High-content screen for modifiers of Niemann-Pick type C disease in patient cells.

Niemann-Pick type C (NPC) disease is a rare lysosomal storage disease caused primarily by mutations in NPC1. NPC1 encodes the lysosomal cholesterol transport protein NPC1. The most common NPC1 mutation is a missense mutation (NPC1I1061T) that causes misfolding and rapid degradation of mutant protein in the endoplasmic reticulum. Cholesterol accumulates in enlarged lysosomes as a result of decreased levels of lysosomal NPC1I1061T protein in patient cells. There is currently no cure or FDA-approved treatment for patients. We sought to identify novel compounds that decrease lysosomal cholesterol storage in NPC1I1061T/I1061T patient fibroblasts using a high-content screen with the cholesterol dye, filipin and the lysosomal marker, LAMP1. A total of 3532 compounds were screened, including 2013 FDA-approved drugs, 327 kinase inhibitors and 760 serum metabolites. Twenty-three hits were identified that decreased both filipin and LAMP1 signals. The majority of hits (16/21) were histone deacetylase (HDAC) inhibitors, a previously described class of modifiers of NPC cholesterol storage. Of the remaining hits, the antimicrobial compound, alexidine dihydrochloride had the most potent lysosomal cholesterol-reducing activity. Subsequent analyses showed that alexidine specifically increased levels of NPC1 transcript and mature protein in both control and NPC patient cells. Although unsuitable for systemic therapy, alexidine represents a unique tool compound for further NPC studies and as a potent inducer of NPC1. Together, these findings confirm the utility of high-content image-based compound screens of NPC1 patient cells and support extending the approach into larger compound collections.

A Novel Flavonoid Glucoside from the Fruits of Lycium ruthenicun.

A novel flavonoid glucoside, ruthenicunoid A (1), together with eight known substances, were isolated from the fruits of Lycium ruthenicun Murr. Their structures were elucidated by extensive spectroscopic data and chemical methods. Especially, the absolute configuration of glucose residue in 1 was assigned by acid hydrolysis followed by derivatization and GC analysis. Biological evaluation towards Sirtuin 1 (SIRT1) found that compounds 1 and 2 exhibit inhibitory activity against SIRT1 in a concentration-dependent manner, indicating its potential on SIRT1-associated disorders.

The Fungal Metabolite Eurochevalierine, a Sequiterpene Alkaloid, Displays Anti-Cancer Properties through Selective Sirtuin 1/2 Inhibition.

NAD⁺-dependent histone deacetylases (sirtuins) are implicated in cellular processes such as proliferation, DNA repair, and apoptosis by regulating gene expression and the functions of numerous proteins. Due to their key role in cells, the discovery of small molecule sirtuin modulators has been of significant interest for diverse therapeutic applications. In particular, it has been shown that inhibition of sirtuin 1 and 2 activities is beneficial for cancer treatment. Here, we demonstrate that the fungal metabolite eurochevalierine from the fungus Neosartorya pseudofischeri inhibits sirtuin 1 and 2 activities (IC50 about 10 µM) without affecting sirtuin 3 activity. The binding modes of the eurochevalierine for sirtuin 1 and 2 have been identified through computational docking analyses. Accordingly, this sequiterpene alkaloid induces histone H4 and α-tubulin acetylation in various cancer cell models in which it induces strong cytostatic effects without affecting significantly the viability of healthy PBMCs. Importantly, eurochevalierine targets preferentially cancer cell proliferation (selectivity factor ≫ 7), as normal human primary CD34⁺ stem/progenitor cells were less affected by the treatment. Finally, eurochevalierine displays suitable drug-likeness parameters and therefore represent a promising scaffold for lead molecule optimization to study the mechanism and biological roles of sirtuins and potentially a basis for development into therapeutics.