A Novel High Content Imaging-Based Screen Identifies the Anti-Helminthic Niclosamide as an Inhibitor of Lysosome Anterograde Trafficking and Prostate Cancer Cell Invasion

Lysosome trafficking plays a significant role in tumor invasion, a key event for the development of metastasis. Previous studies from our laboratory have demonstrated that the anterograde (outward) movement of lysosomes to the cell surface in response to certain tumor microenvironment stimulus, such as hepatocyte growth factor (HGF) or acidic extracellular pH (pHe), increases cathepsin B secretion and tumor cell invasion. Anterograde lysosome trafficking depends on sodium-proton exchanger activity and can be reversed by blocking these ion pumps with Troglitazone or EIPA. Since these drugs cannot be advanced into the clinic due to toxicity, we have designed a high-content assay to discover drugs that block peripheral lysosome trafficking with the goal of identifying novel drugs that inhibit tumor cell invasion. An automated high-content imaging system (Cellomics) was used to measure the position of lysosomes relative to the nucleus. Among a total of 2210 repurposed and natural product drugs screened, 18 “hits” were identified. One of the compounds identified as an anterograde lysosome trafficking inhibitor was niclosamide, a marketed human anti-helminthic drug. Further studies revealed that niclosamide blocked acidic pHe, HGF, and epidermal growth factor (EGF)-induced anterograde lysosome redistribution, protease secretion, motility, and invasion of DU145 castrate resistant prostate cancer cells at clinically relevant concentrations. In an effort to identify the mechanism by which niclosamide prevented anterograde lysosome movement, we found that this drug exhibited no significant effect on the level of ATP, microtubules or actin filaments, and had minimal effect on the PI3K and MAPK pathways. Niclosamide collapsed intralysosomal pH without disruption of the lysosome membrane, while bafilomycin, an agent that impairs lysosome acidification, was also found to induce JLA in our model. Taken together, these data suggest that niclosamide promotes juxtanuclear lysosome aggregation (JLA) via modulation of pathways involved in lysosome acidification. In conclusion, we have designed a validated reproducible high-content assay to screen for drugs that inhibit lysosome trafficking and reduce tumor invasion and we summarize the action of one of these drugs.     

Synergistic interactions between HDAC and sirtuin inhibitors in human leukemia cells

Aberrant histone deacetylase (HDAC) activity is frequent in human leukemias. However, while classical, NAD(+)-independent HDACs are an established therapeutic target, the relevance of NAD(+)-dependent HDACs (sirtuins) in leukemia treatment remains unclear. Here, we assessed the antileukemic activity of sirtuin inhibitors and of the NAD(+)-lowering drug FK866, alone and in combination with traditional HDAC inhibitors. Primary leukemia cells, leukemia cell lines, healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol, cambinol, EX527) and with FK866, with or without addition of the HDAC inhibitors valproic acid, sodium butyrate, and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis, and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD(+) levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells, but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells, HDAC inhibitors were found to induce upregulation of Bax, a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result, leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion, NAD(+)-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited.     

Thiazolidinediones Induce Rab7–RILP–MAPK‐Dependent Juxtanuclear Lysosome Aggregation and Reduce Tumor Cell Invasion

Acidic extracellular pH (pHe) has been shown to stimulate peripheral lysosome trafficking, resulting in cathepsin B secretion and tumor invasion. In addition, inhibitors of sodium‐proton exchangers (NHE) such as EIPA, cariporide and s3226, as well as the non‐specific NHE inhibitor, troglitazone (Tro), blocked these changes. In this paper, we report a differential ability of the thiazolidinedione (TZD) family of compounds to induce a time‐dependent retrograde aggregation of lysosomes over the microtubule‐organizing center (MTOC) in tumor cells exposed to acidic pHe. This trafficking event depended on microtubules and the MAP‐Kinase pathway, but was independent of Rho GTPase activity. Expression of shRNA implicated Rab7 in this process, and subcellular fractionation revealed that levels of Rab7, RILP and Erk1/2 were increased on lysosomes purified from cells treated with Tro. In addition, DN‐RILP overexpression studies indicated that this Rab7 effector also played a role in TZD‐induced retrograde trafficking. Tro was able to prevent acidic pHe‐induced cell invasion. Finally, DU145 prostate tumor cells stably over‐expressing WT‐RILP, a condition where lysosomes aggregate to the MTOC in the absence of Tro, did not invade in response to acidic pHe, suggesting that the regulation of lysosome trafficking is an inherently important aspect of tumor cell invasion.     

Inhibition of the NAD-Dependent Protein Deacetylase SIRT2 Induces Granulocytic Differentiation in Human Leukemia Cells

Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL) cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia–retinoic acid receptor α (PML-RAR-α) stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation.     

Simplification of the tetracyclic SIRT1-selective inhibitor MC2141: coumarin- and pyrimidine-based SIRT1/2 inhibitors with different selectivity profile

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.     

Sirtuin 1 (SIRT1): the misunderstood HDAC

The sirtuin family of NAD-dependent histone deacetylases (HDACs) consists of seven mammalian proteins, SIRT1-7. Many of the sirtuin isoforms also deacetylate nonhistone substrates, such as p53 (SIRT1) and α-tubulin (SIRT2). The sirtuin literature focuses on pharmacological activators of SIRT1 (e.g., resveratrol, SRT1720), proposed as therapeutics for diabetes, neurodegeneration, inflammation, and others. However, many of the SIRT1 activator results may have been due to artifacts in the assay methodology (i.e., use of fluorescently tagged substrates). A biological role for SIRT1 in cancer has been given less scrutiny but is no less equivocal. Although proposed initially as an oncogene, we present herein compelling data suggesting that SIRT1 is indeed a context-specific tumor suppressor. For oncology, SIRT1 inhibitors (dual SIRT1/2) are indicated as potential therapeutics. A number of sirtuin inhibitors have been developed but with mixed results in cellular systems and animal models. It is unclear whether this has been due to poorly understood model systems, signalling redundancy, and/or inadequately potent and selective tool compounds. This review provides an overview of recent developments in the field of SIRT1 function. While focusing on oncology, it aims to shed light on new concepts of expanding the selectivity spectrum, including other sirtuins such as SIRT2.     

Unexpected small molecules as novel SIRT2 suicide inhibitors

A series of sirtuin inhibitor candidates were assembled based on an intermediate ester (1a) our accidently discovered. After screening and evaluation, several SIRT2 selective inhibitors were identified, which can inhibit all the deacetylation, defatty-acylation and debenzoylation of SIRT2. Among these inhibitors, compound 1e was the best SIRT2 selective inhibitors. The primary study on the inhibitory mechanism indicated that compound 1e may be a suicide inhibitor acting as an irreversible way. Given almost all reported sirtuin inhibitors are non-covalent, sirtuin covalent inhibitors are still need to be developed. These findings will facilitate for further development of SIRT2 selective and suicide inhibitors.     

SIRT1 negatively regulates the protein stability of HIPK2

In the present study, we investigated whether a histone deacetylase sirtuin 1 (SIRT1) can regulate the protein stability of homeodomain-interacting protein kinase 2 (HIPK2). We observed the evidence of molecular interaction between SIRT1 and HIPK2. Interestingly, overexpression or pharmacological activation of SIRT1 promoted ubiquitination and the proteasomal degradation of HIPK2 whereas inhibition of SIRT1 activity increased the protein level of HIPK2. Furthermore, a SIRT1 activator decreased the level of HIPK2 acetylation whereas an inhibitor increased the acetylation level. These results suggest that SIRT1 may deacetylate and promote the ubiquitination and subsequent proteasomal degradation of HIPK2.     

A novel role of SIRT2 in regulating gap junction communications via connexin-43 in bovine cumulus-oocyte complexes

SIRT2, the predominantly cytosolic sirtuin, plays important role in multiple biological processes, including metabolism, stress response, and aging. However, the function of SIRT2 in gap junction intercellular communications (GJICs) of cumulus-oocyte complexes (COCs) is not yet known. The purpose of the present study was to evaluate the effect and underlining mechanism of SIRT2 on GJICs in COCs. Here, we found that treatment with SIRT2 inhibitors (SirReal2 or TM) inhibited bovine oocyte nuclear maturation. Further analysis revealed that SIRT2 inactivation disturbed the GJICs of COCs during in vitro maturation. Correspondingly, both the Cx43 phosphorylation levels and MEK/MER signaling pathways were induced by SIRT2 inhibition. Importantly, SIRT2-mediated Cx43 phosphorylation was completely abolished by treatment with MEK1/2 inhibitor (Trametinib). Furthermore, treatment with SIRT2 inhibitors resulted in the high levels of MEK1/2 acetylation. Functionally, downregulating the MER/ERK pathways with inhibitors (Trametinib or SCH772984) could attenuate the closure of GJICs caused by SIRT2 inactivation in partly. In addition, inhibition of SIRT2 activity significantly decreased the membrane and zona pellucida localization of Cx43 by upregulating the levels of Cx43 acetylation. Taken together, these results demonstrated a novel role that SIRT2 regulates GJICs via modulating the phosphorylation and deacetylation of Cx43 in COCs.     

Cambinol,a Novel Inhibitor of Neutral Sphingomyelinase 2 Shows Neuroprotective Properties

Ceramide is a bioactive lipid that plays an important role in stress responses leading to apoptosis, cell growth arrest and differentiation. Ceramide production is due in part to sphingomyelin hydrolysis by sphingomyelinases. In brain, neutral sphingomyelinase 2 (nSMase2) is expressed in neurons and increases in its activity and expression have been associated with pro-inflammatory conditions observed in Alzheimer’s disease, multiple sclerosis and human immunodeficiency virus (HIV-1) patients. Increased nSMase2 activity translates into higher ceramide levels and neuronal cell death, which can be prevented by chemical or genetic inhibition of nSMase2 activity or expression. However, to date, there are no soluble, specific and potent small molecule inhibitor tool compounds for in vivo studies or as a starting point for medicinal chemistry optimization. Moreover, the majority of the known inhibitors were identified using bacterial, bovine or rat nSMase2. In an attempt to identify new inhibitor scaffolds, two activity assays were optimized as screening platform using the recombinant human enzyme. First, active hits were identified using a fluorescence-based high throughput compatible assay. Then, hits were confirmed using a ¹⁴C sphingomyelin-based direct activity assay. Pharmacologically active compounds and approved drugs were screened using this strategy which led to the identification of cambinol as a novel uncompetitive nSMase2 inhibitor (K_i = 7 μM). The inhibitory activity of cambinol for nSMase2 was approximately 10-fold more potent than for its previously known target, silence information regulator 1 and 2 (SIRT1/2). Cambinol decreased tumor necrosis factor-α or interleukin-1 β-induced increases of ceramide and cell death in primary neurons. A preliminary study of cambinol structure and activity allowed the identification of the main structural features required for nSMase2 inhibition. Cambinol and its analogs may be useful as nSMase2 inhibitor tool compounds to prevent ceramide-dependent neurodegeneration.     

Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome

Autophagy protects organelles, cells, and organisms against several stress conditions. Induction of autophagy by resveratrol requires the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1). In this paper, we show that the acetylase inhibitor spermidine stimulates autophagy independent of SIRT1 in human and yeast cells as well as in nematodes. Although resveratrol and spermidine ignite autophagy through distinct mechanisms, these compounds stimulate convergent pathways that culminate in concordant modifications of the acetylproteome. Both agents favor convergent deacetylation and acetylation reactions in the cytosol and in the nucleus, respectively. Both resveratrol and spermidine were able to induce autophagy in cytoplasts (enucleated cells). Moreover, a cytoplasm-restricted mutant of SIRT1 could stimulate autophagy, suggesting that cytoplasmic deacetylation reactions dictate the autophagic cascade. At doses at which neither resveratrol nor spermidine stimulated autophagy alone, these agents synergistically induced autophagy. Altogether, these data underscore the importance of an autophagy regulatory network of antagonistic deacetylases and acetylases that can be pharmacologically manipulated.     

Evaluation of benzoic acid derivatives as sirtuin inhibitors

Employing a genetically modified yeast strain as a screening tool, 4-dimethylaminobenzoic acid (5) was isolated from the marine sediment-derived Streptomyces sp. CP27-53 as a weak yeast sirtuin (Sir2p) inhibitor. Using this compound as a scaffold, a series of disubstituted benzene derivatives were evaluated to elucidate the structure activity relationships for Sir2p inhibition. The results suggested that 4-alkyl or 4-alkylaminobenzoic acid is the key structure motif for Sir2p inhibitory activity. The most potent Sir2p inhibitor, 4-tert-butylbenzoic acid (20), among the tested compounds in this study turned out to be a weak but selective SIRT1 inhibitor. The calculated binding free energies between the selected compounds and the catalytic domain of SIRT1 were well correlated to their measured SIRT1 inhibitory activities.     

SIRT1 contains N- and C-terminal regions that potentiate deacetylase activity

SIRT1 is one of seven mammalian sirtuin (silent information regulator 2-related) proteins that harbor NAD(+)-dependent protein deacetylase activity and is implicated in multiple metabolic and age-associated pathways and disorders. The sirtuin proteins contain a central region of high sequence conservation that is required for catalytic activity, but more variable N- and C-terminal regions have been proposed to mediate protein specific activities. Here we show that the conserved catalytic core domain of SIRT1 has very low catalytic activity toward several known protein substrates, but that regions N- and C-terminal to the catalytic core potentiate catalytic efficiency by between 12- and 45-fold, with the N-terminal domain contributing predominantly to catalytic rate, relatively independent of the nature of the acetyl-lysine protein substrate, and the C-terminal domain contributing significantly to the K(m) for NAD(+). We show that the N- and C-terminal regions stimulate SIRT1 deacetylase activity intramolecularly and that the C-terminal region stably associates with the catalytic core domain to form a SIRT1 holoenzyme. We also demonstrate that the C-terminal region of SIRT1 can influence the inhibitory activity of some sirtuin inhibitors that are known to function through the catalytic core domain. Together, these studies highlight the unique properties of the SIRT1 member of the sirtuin proteins and have implications for the development of SIRT1-specific regulatory molecules.     

Inhibition of Glioma Cell Lysosome Exocytosis Inhibits Glioma Invasion

Cancer cells invade by secreting enzymes that degrade the extracellular matrix and these are sequestered in lysosomal vesicles. In this study, the effects of the selective lysosome lysing drug GPN and the lysosome exocytosis inhibitor vacuolin-1 on lysosome exocytosis were studied to determine their effect on glioma cell migration and invasion. Both GPN and vacuolin-1 evidently inhibited migration and invasion in transwell experiments and scratch experiments. There are more lysosomes located on the cell membrane of glioma cells than of astrocytes. GPN decreased the lysosome number on the cell membrane. We found that rab27A was expressed in glioma cells, and colocalized with cathepsin D in lysosome. RNAi-Rab27A inhibited lysosome cathepsin D exocytosis and glioma cell invasion in an in vitro assay. Inhibition of cathepsin D inhibited glioma cell migration. The data suggest that the inhibition of lysosome exocytosis from glioma cells plays an important modulatory role in their migration and invasion.     

After the grape rush: sirtuins as epigenetic drug targets in neurodegenerative disorders

Class III histone deacetylases (sirtuins) are becoming increasingly recognized as important epigenetic drug targets in cancer and metabolic disorders. As key regulators involved in numerous cellular signalling pathways, sirtuins are also emerging as potential targets in various neurodegenerative diseases such as Alzheimer, Parkinson's disease and others, thus suggesting modulation of sirtuin activity could provide an interesting and novel therapeutic option. In particular, much attention has been raised by neuroprotective effects attributed to SIRT1 activation due to genetically induced sirtuin overexpression or administration of resveratrol, a natural compound found in the skin of red grapes and also in wine. Similarly, also sirtuin inhibitors display benefits in various neuropathologic disease models. In light of the growing interest in sirtuin modulation and with regard to the lack of conclusive data on small molecule activators of sirtuins this review recapitulates the known facts about sirtuins and their relevance in neurodegenerative diseases.