Is schedule IV suvorexant an appropriate reference drug of abuse to use in preclinical abuse liability testing in the rat?

The abuse potential of novel CNS-active drug candidates with low specificity for known receptors involved in abuse might be complex to test preclinically relative to an appropriate reference drug of abuse. Suvorexant, a Schedule IV dual orexin receptor antagonist was investigated for its potential use as a reference drug in Drug Discrimination Learning (DDL) studies. Firstly, toxicokinetic properties of suvorexant were determined in male and female rats after single oral doses of 160 and 325 mg/kg in MC and PEG400. Thereafter the subjective effects of suvorexant at 325 mg/kg versus vehicle were evaluated in a DDL paradigm and plasma exposures were measured. Mean maximum plasma exposures in male rats after a single dose of 325 mg/kg suvorexant were 2.5- (MC) to 10.5-fold (PEG400) the human exposure at supratherapeutic doses of 40 mg q.d. (Cmax:1.1 µM), and 4.9- (MC) to 20.8-fold (PEG400) the approved maximum human efficacious dose (20 mg q.d.; 0.557 µM). Training male rats at 325 mg/kg in the DDL study however did not result in discriminative stimulus generalisation versus respective vehicles. Suvorexant, a Schedule IV dual orexin receptor antagonist failed to serve as a robust reference drug of abuse in the DDL paradigm in rats despite appropriate exposures.

Synthesis, antibacterial and antifungal activity of new 3-biphenyl-3H-Imidazo[1,2-a]azepin-1-ium bromides.

Novel 1-aryl-3-biphenyl-4-yl-3-hydroxy-2,5,6,7,8,9-hexahydro-3H-imidazo[1,2-a]azepin-1-ium bromides and their 2,5-dehydrogenated analogues were designed and synthesized using a reaction of aryl-(4,5,6,7-tetrahydro-3H-azepin-2-yl)-amines with 1-biphenyl-4-yl-2-bromoethanone. Among the 16 novel compounds 5 derivatives displayed in vitro antimicrobial activity; while three of them showed promising activity against Staphylococcus aureus, Cryptococcus neoformans and Candida albicans.

Chronic activation of FXR-induced liver growth with tissue-specific targeting Cyclin D1.

The nuclear receptor (FXR) plays essential roles in maintaining bile acid and lipid homeostasis by regulating diverse target genes. And its agonists were promising agents for treating various liver diseases. Nevertheless, the potential side effect of chronic FXR activation by specific agonists is not fully understood. In this study, we investigated the mechanism of FXR agonist WAY-362450 induced liver enlargement during treating liver diseases. We demonstrated that chronic ingestion of WAY-362450 induced liver hypertrophy instead of hyperplasia in mouse. Global transcriptional pattern was also examined in mouse livers after treatment with WAY-362450 by RNA-seq assay. Through GO and KEGG enrichment analyses, we demonstrated that the expression of Cyclin D1 (Ccnd1) among the cell cycle-regulating genes was notably increased in WAY-362450-treated mouse liver. Activation of FXR-induced Ccnd1 expression in hepatocyte in a time-dependent manner in vivo and in vitro. Through bioinformatics analysis and ChIP assay, we identified FXR as a direct transcriptional activator of Ccnd1 through binding to a potential enhancer, which was specifically active in livers. We also found active histone acetylation was essential for Ccnd1 induction by FXR. Thus, our study indicated that activation of FXR-induced harmless liver hypertrophy with spatiotemporal modulation of Ccnd1. With a better understanding of the mechanism of tissue-specific gene regulation by FXR, it is beneficial for development and appropriate application of its specific agonist in preventing hepatic diseases.

Study of degradation behavior of besifloxacin, characterization of its degradation products by LC-ESI-QTOF-MS and their in silico toxicity prediction.

Knowledge and understanding of the stability profile of a drug is important as it affects its safety and efficacy. In the present work, besifloxacin, a new, fourth-generation fluoroquinolone antibiotic, was subjected to different forced-degradation conditions as per International Conference on Harmonization (ICH) guidelines such as hydrolysis (acid, base and neutral), oxidation, thermal and photolysis. The drug degraded under acidic, basic, oxidative and photolytic conditions while it was found to be stable under dry heat and neutral hydrolytic conditions. In total, five degradation products (DPs) were formed under different conditions-DP1 and DP2 (photolysis), DP3 (oxidation), DP4 (acidic), DP3 and DP5 (basic). The chromatographic separation of besifloxacin and its degradation products was achieved on a Sunfire C18 (250 mm × 4.6 mm, 5 µm) column with 0.1% aqueous formic acid-acetonitrile as a mobile phase. The gradient RP-HPLC method was developed and validated as per ICH guidelines. The degradation products were characterized with the help of LC-ESI-QTOF mass spectrometric studies and the most likely degradation pathway of the drug was proposed. In silico toxicity assessment of the drug and its degradation products was carried out, which indicated that DP3 and DP4 carry a mutagenicity alert.

Novel naphthyloxy derivatives - Potent histamine H3 receptor ligands. Synthesis and pharmacological evaluation.

A series of 1- and 2-naphthyloxy derivatives were synthesized and evaluated for histamine H3 receptor affinity. Most compounds showed high affinities with Ki values below 100 nM. The most potent ligand, 1-(5-(naphthalen-1-yloxy)pentyl)azepane (11) displayed high affinity for the histamine H3 receptor with a Ki value of 21.9 nM. The antagonist behaviour of 11 was confirmed both in vitro in the cAMP assay (IC50 = 312 nM) and in vivo in the rat dipsogenia model (ED50 = 3.68 nM). Moreover, compound 11 showed positive effects on scopolamine induced-memory deficits in mice (at doses of 10 and 15 mg/kg) and an analgesic effect in the formalin test in mice with ED50 = 30.6 mg/kg (early phase) and ED50 = 20.8 mg/kg (late phase). Another interesting compound, 1-(5-(Naphthalen-1-yloxy)pentyl)piperidine (13; H3R Ki = 53.9 nM), was accepted for Anticonvulsant Screening Program at the National Institute of Neurological Disorders and Stroke/National Institute of Health (Rockville, USA). The screening was performed in the maximal electroshock seizure (MES), the subcutaneous pentylenetetrazole (scPTZ) and the 6-Hz psychomotor animal models of epilepsy. Neurologic deficit was evaluated by the rotarod test. Compound 13 inhibited convulsions induced by the MES with ED50 of 19.2 mg/kg (mice, i.p.), 17.8 (rats, i.p.), and 78.1 (rats, p.o.). Moreover, 13 displayed protection against the 6-Hz psychomotor seizures (32 mA) in mice (i.p.) with ED50 of 33.1 mg/kg and (44 mA) ED50 of 57.2 mg/kg. Furthermore, compounds 11 and 13 showed in vitro weak influence on viability of tested cell lines (normal HEK293, neuroblastoma IMR-32, hepatoma HEPG2), weak inhibition of CYP3A4 activity, and no mutagenicity. Thus, these compounds may be used as leads in a further search for histamine H3 receptor ligands with promising in vitro and in vivo activity.

The STIM1 inhibitor ML9 disrupts basal autophagy in cardiomyocytes by decreasing lysosome content.

Stromal-interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) plays a key role in mediating cardiomyocyte hypertrophy, both in vitro and in vivo. Moreover, there is growing support for the contribution of SOCE to the Ca2+ overload associated with ischemia/reperfusion injury. Therefore, STIM1 inhibition is proposed as a novel target for controlling both hypertrophy and ischemia/reperfusion-induced Ca2+ overload. Our aim was to evaluate the effect of ML9, a STIM1 inhibitor, on cardiomyocyte viability. ML9 was found to induce cell death in cultured neonatal rat cardiomyocytes. Caspase-3 activation, apoptotic index and release of the necrosis marker lactate dehydrogenase to the extracellular medium were evaluated. ML9-induced cardiomyocyte death was not associated with increased intracellular ROS or decreased ATP levels. Moreover, treatment with ML9 significantly increased levels of the autophagy marker LC3-II, without altering Beclin1 or p62 protein levels. However, treatment with ML9 followed by bafilomycin-A1 did not produce further increases in LC3-II content. Furthermore, treatment with ML9 resulted in decreased LysoTracker® Green staining. Collectively, these data suggest that ML9-induced cardiomyocyte death is triggered by a ML9-dependent disruption of autophagic flux due to lysosomal dysfunction.

Indoloazepinone-Constrained Oligomers as Cell-Penetrating and Blood-Brain-Barrier-Permeating Compounds.

Non-cationic and amphipathic indoloazepinone-constrained (Aia) oligomers have been synthesized as new vectors for intracellular delivery. The conformational preferences of the [l-Aia-Xxx]n oligomers were investigated by circular dichroism (CD) and NMR spectroscopy. Whereas Boc-[l-Aia-Gly]2,4 -OBn oligomers 12 and 13 and Boc-[l-Aia-ß3 -h-l-Ala]2,4 -OBn oligomers 16 and 17 were totally or partially disordered, Boc-[l-Aia-l-Ala]2 -OBn (14) induced a typical turn stabilized by C5 - and C7 -membered H-bond pseudo-cycles and aromatic interactions. Boc-[l-Aia-l-Ala]4 -OBn (15) exhibited a unique structure with remarkable T-shaped π-stacking interactions involving the indole rings of the four l-Aia residues forming a dense hydrophobic cluster. All of the proposed FITC-6-Ahx-[l-Aia-Xxx]4 -NH2 oligomers 19-23, with the exception of FITC-6-Ahx-[l-Aia-Gly]4 -NH2 (18), were internalized by MDA-MB-231 cells with higher efficiency than the positive references penetratin and Arg8 . In parallel, the compounds of this series were successfully explored in an in vitro blood-brain barrier (BBB) permeation assay. Although no passive diffusion permeability was observed for any of the tested Ac-[l-Aia-Xxx]4 -NH2 oligomers in the PAMPA model, Ac-[l-Aia-l-Arg]4 -NH2 (26) showed significant permeation in the in vitro cell-based human model of the BBB, suggesting an active mechanism of cell penetration.

Contrasting effect of the latency-reversing agents bryostatin-1 and JQ1 on astrocyte-mediated neuroinflammation and brain neutrophil invasion.

BACKGROUND: Despite effectiveness of the combined antiretroviral therapy, HIV-1 persists in long-lived latently infected cells. Consequently, new therapeutic approaches aimed at eliminating this latent reservoir are currently being developed. A "shock and kill" strategy using latency-reversing agents (LRA) to reactivate HIV-1 has been proposed. However, the impact of LRA on the central nervous system (CNS) remains elusive. METHODS: We used human fetal astrocytes and investigated the effects of several LRA on their functional and secretory activities. Astrocytes were infected with VSV-G-pseudotyped HIV-1 before treatment with various blood-brain barrier (BBB)-permeable LRA at subcytotoxic doses, which allow HIV-1 reactivation based on previous in vitro and clinical studies. Cells and supernatants were then used to evaluate effects of infection and LRA on (i) viability and metabolic activity of astrocytes using a colorimetric MTS assay; (ii) chemokines and proinflammatory cytokines secretion and gene expression by astrocytes using ELISA and RT-qPCR, respectively; (iii) expression of complement component 3 (C3), a proxy for astrogliosis, by RT-qPCR; (iv) glutamate uptake capacity by a fluorometric assay; and (v) modulation of neutrophil transmigration across an in vitro BBB model. RESULTS: We demonstrate that bryostatin-1 induces secretion of chemokines CCL2 and IL-8 and proinflammatory cytokines IL-6 and GM-CSF, whereas their production is repressed by JQ1. Bryostatin-1 also increases expression of complement component 3 and perturbs astrocyte glutamate homeostasis. Lastly, bryostatin-1 enhances transmigration of neutrophils across an in vitro blood-brain barrier model and induces formation of neutrophil extracellular traps. CONCLUSIONS: These observations highlight the need to carefully assess the potential harmful effect to the CNS when selecting LRA for HIV-1 reactivation strategies.

Chiral-pool synthesis of 1,2,4-trisubstituted 1,4-diazepanes as novel σ1 receptor ligands.

Starting from enantiomerically pure amino acids, 1,4-diazepanes with various substituents in 1, 2, and 4-position were synthesized following the late stage diversification strategy. The key step in the formation of the seven-membered ring was the intramolecular EDC coupling of amino acids 15, 26, and 39. The configuration in 2-position does not influence the σ1 affinity and selectivity over related receptors. A cyclohexylmethyl or a butyl group are the preferred substituents in 4-position, whereas a methyl moiety in 2-position and a (substituted) benzyl moiety in 1-position result in the highest σ1 affinity. These results fit nicely to the reported σ1 pharmacophore models. The compounds did not inhibit the structurally related fungal enzyme sterol Δ8,7-isomerase, but showed inhibition of diverse enzymes in late cholesterol biosynthesis at high concentrations. In a screening against more than 50 target proteins, (2S)-1-benzyl-4-(4-methoxybenzyl)-2-methyl-1,4-diazepane ((S)-28b, Ki(σ1)=0.86nM) showed a clean receptor profile. The dose dependent potentiation of electrically stimulated contractions of guinea pig vas deferens indicates σ1 agonistic activity of (S)-28b. Even at a dose of 100mg/kg (S)-28b did not induce severe toxic or behavioral effects in the Irwin screen. Clear cognition enhancing effects were observed for (S)-28b after inducing amnesia by scopolamine.

SM-1, a novel PAC-1 derivative, activates procaspase-3 and causes cancer cell apoptosis.

PURPOSE: To develop more potent procaspase-3 activator, 7 novel derivatives of PAC-1 were synthesized and evaluated. Among them, SM-1 stood out for its promising activity and good pharmacokinetics properties. The purpose of this study is to elucidate the pharmacological mechanism of SM-1 and evaluate its efficacy and toxicity in-depth. METHODS: To reveal the effects of SM-1 on caspase-3 activity, both in vitro activation assay and in cells fluorometric assay were tested. The protein levels and distributions of procaspase-3 and cleaved caspase-3 were also measured by western blot and immunostaining. MTT assay, apoptosis assay and mouse xenograft model were applied to evaluate the efficacy of SM-1. Preliminary safety assessments also tested the acute toxicity and tissue distribution of SM-1. RESULTS: Compared to PAC-1, SM-1 showed higher cytotoxicity in cancer cells. Further investigation demonstrated that SM-1 relieved zinc-mediated inhibition of procaspase-3 and activated the caspase-3 activity both in tube test and in cells. Efficacy evaluation showed SM-1-induced cell apoptosis mainly via activation of caspase-3 and reduced tumor size in mouse xenograft model. Its apoptosis induction efficacy was higher than PAC-1. The preliminary safety assessment demonstrated that the overall LD50 of SM-1 lied between 500 and 1000 mg/kg and the distribution of SM-1 in brain was low. CONCLUSIONS: We identified SM-1 as a promising antitumor candidate, which displayed enhanced procaspase-3 activating activity and potent cytotoxicity for cancer cells but low toxicity for normal cells.

Toxicity of Intracameral Injection of Fourth-Generation Fluoroquinolones on the Corneal Endothelium.

PURPOSE: The aim of this study was to compare the cellular susceptibility patterns and morphologic changes in the corneal endothelium associated with the use of fourth-generation fluoroquinolones. METHOD: Endothelial susceptibility was assessed through intracameral injection of besifloxacin, gatifloxacin, and moxifloxacin. Human umbilical vein endothelial cells (HUVECs) were used as the standard cellular lineage to assess the quantitative toxicity of each antibiotic solution. Qualitative changes in the morphologic character of the corneal structure and the endothelial layer were generated using a combination of ex vivo and in vivo assays. Experimental assays were conducted in triplicate, and the results were statistically analyzed. RESULTS: At 1 hour of exposure, all HUVECs exposed to antibiotics showed viability above 85%, after 3 hours of exposure to besifloxacin, gatifloxacin, and moxifloxacin, the percentages of viable cells were 68.3 ± 4.0 (P < 0.001), 90.7 ± 4.2 (P < 0.05), and 93.3 ± 1.5 (P > 0.05), respectively. All fluoroquinolones tested showed toxicity to HUVECs, resulting in significant (P < 0.001) loss of cellular viability after 24 hours of drug exposure. Giant endothelial cells were observed in animals treated with the 3 fluoroquinolones in contrast to the absence of these abnormal cells in the untreated group. Early cellular detachment was seen in the endothelial layer after exposure to gatifloxacin and moxifloxacin. CONCLUSIONS: We concluded that injection of fourth-generation fluoroquinolones in the aqueous humor did not adversely affect the corneal endothelium. However, these results suggested that prophylactic intracameral injection of besifloxacin, gatifloxacin, or moxifloxacin, if needed, should be administered as a last therapeutic resource in clinical practice, with careful and constant monitoring of corneal endothelium.

ML-7 amplifies the quinocetone-induced cell death through akt and MAPK-mediated apoptosis on HepG2 cell line.

The study aims at evaluating the combination of the quinocetone and the ML-7 in preclinical hepatocellular carcinoma models. To this end, the effect of quinocetone and ML-7 on apoptosis induction and signaling pathways was analyzed on HepG2 cell lines. Here, we report that ML-7, in a nontoxic concentration, sensitized the HepG2 cells to quinocetone-induced cytotoxicity. Also, ML-7 profoundly enhances quinocetone-induced apoptosis in HepG2 cell line. Mechanistic investigations revealed that ML-7 and quinocetone act in concert to trigger the cleavage of caspase-8 as well as Bax/Bcl-2 ratio up-regulation and subsequent cleavage of Bid, capsases-9 and -3. Importantly, ML-7 weakened the quinocetone-induced Akt pathway activation, but strengthened the phosphorylation of p-38, ERK and JNK. Further treatment of Akt activator and p-38 inhibitor almost completely abolished the ML-7/quinocetone-induced apoptosis. In contrast, the ERK and JNK inhibitor aggravated the ML-7/quinocetone-induced apoptosis, indicating that the synergism critically depended on p-38 phosphorylation and HepG2 cells provoke Akt, ERK and JNK signaling pathways to against apoptosis. In conclusion, the rational combination of quinocetone and ML-7 presents a promising approach to trigger apoptosis in hepatocellular carcinoma, which warrants further investigation.

Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4.

BRD4, a bromodomain and extraterminal domain (BET) family member, is an attractive target in multiple pathological settings, particularly cancer. While BRD4 inhibitors have shown some promise in MYC-driven malignancies such as Burkitt's lymphoma (BL), we show that BRD4 inhibitors lead to robust BRD4 protein accumulation, which may account for their limited suppression of MYC expression, modest antiproliferative activity, and lack of apoptotic induction. To address these limitations we designed ARV-825, a hetero-bifunctional PROTAC (Proteolysis Targeting Chimera) that recruits BRD4 to the E3 ubiquitin ligase cereblon, leading to fast, efficient, and prolonged degradation of BRD4 in all BL cell lines tested. Consequently, ARV-825 more effectively suppresses c-MYC levels and downstream signaling than small-molecule BRD4 inhibitors, resulting in more effective cell proliferation inhibition and apoptosis induction in BL. Our findings provide strong evidence that cereblon-based PROTACs provide a better and more efficient strategy in targeting BRD4 than traditional small-molecule inhibitors.

Therapeutic effects of R8, a semi-synthetic analogue of Vasicine, on murine model of allergic airway inflammation via STAT6 inhibition.

This is a follow-up study of our previous work in which we screened a series of Vasicine analogues for their anti-inflammatory activity in a preventive OVA induced murine model of asthma. The study demonstrated that R8, one of the analogues, significantly suppressed the Th2 cytokine production and eosinophil recruitment to the airways. In the present study, we have been using two standard experimental murine models of asthma, where the mice were treated with R8 either during (preventive use) or after (therapeutic use) the development of asthma features. In the preventive model, R8 reduced inflammatory cell infiltration to the airways, OVA specific IgE and Th2 cytokine production. Also, the R8 treatment in the therapeutic model decreased methacholine induced AHR, Th2 cytokine release, serum IgE levels, infiltration of inflammatory cells into the airways, phosphorylation of STAT6 and expression of GATA3. Moreover, R8 not only reduced goblet cell metaplasia in asthmatic mice but also reduced IL-4 induced Muc5AC gene expression in human alveolar basal epithelial cells. Further, R8 attenuated IL-4 induced differentiation of murine splenocytes into Th2 cells in vitro. So, we may deduce that R8 treatment profoundly reduced asthma features by attenuating the differentiation of T cells into Th2 cells by interfering with the binding of IL-4 to its receptor in turn decreasing the phosphorylation of STAT6 and expression of GATA3 in murine model of asthma. These preclinical findings suggest a possible therapeutic role of R8 in allergic asthma.

Molecular targeting of the oncoprotein PLK1 in pediatric acute myeloid leukemia: RO3280, a novel PLK1 inhibitor, induces apoptosis in leukemia cells.

Polo-like kinase 1 (PLK1) is highly expressed in many cancers and therefore a biomarker of transformation and potential target for the development of cancer-specific small molecule drugs. RO3280 was recently identified as a novel PLK1 inhibitor; however its therapeutic effects in leukemia treatment are still unknown. We found that the PLK1 protein was highly expressed in leukemia cell lines as well as 73.3% (11/15) of pediatric acute myeloid leukemia (AML) samples. PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35; p < 0.001). Kaplan-Meier survival analysis revealed that shorter survival time correlated with high tumor PLK1 expression (p = 0.002). The 50% inhibitory concentration (IC50) of RO3280 for acute leukemia cells was between 74 and 797 nM. The IC50 of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was between 35.49 and 110.76 nM and 52.80 and 147.50 nM, respectively. RO3280 induced apoptosis and cell cycle disorder in leukemia cells. RO3280 treatment regulated several apoptosis-associated genes. The regulation of DCC, CDKN1A, BTK, and SOCS2 was verified by western blot. These results provide insights into the potential use of RO3280 for AML therapy; however, the underlying mechanisms remain to be determined.

The selective activation of p53 target genes regulated by SMYD2 in BIX-01294 induced autophagy-related cell death.

Transcription regulation emerged to be one of the key mechanisms in regulating autophagy. Inhibitors of H3K9 methylation activates the expression of LC3B, as well as other autophagy-related genes, and promotes autophagy process. However, the detailed mechanisms of autophagy regulated by nuclear factors remain elusive. In this study, we performed a drug screen of SMYD2-/- cells and discovered that SMYD2 deficiency enhanced the cell death induced by BIX01294, an inhibitor of histone H3K9 methylation. BIX-01294 induces accumulation of LC3 II and autophagy-related cell death, but not caspase-dependent apoptosis. We profiled the global gene expression pattern after treatment with BIX-01294, in comparison with rapamycin. BIX-01294 selectively activates the downstream genes of p53 signaling, such as p21 and DOR, but not PUMA, a typical p53 target gene inducing apoptosis. BIX-01294 also induces other autophagy-related genes, such as ATG4A and ATG9A. SMYD2 is a methyltransferase for p53 and regulates its transcription activity. Its deficiency enhances the BIX-01294-induced autophagy-related cell death through transcriptionally promoting the expression of p53 target genes. Taken together, our data suggest BIX-01294 induces autophagy-related cell death and selectively activates p53 target genes, which is repressed by SMYD2 methyltransferase.

Thee-component, one-pot synthesis of hexahydroazepino[3,4-b]indole and tetrahydro-1H-pyrido[3,4-b]indole derivatives and evaluation of their cytotoxicity.

A three-component, four-center Ugi reaction has been developed to produce a novel class of 2-aryl-3-oxo-hexahydroazepino[3,4-b]indole and 2-aryl-3-oxo-tetrahydro-1H-pyrido[3,4-b]indole derivatives in good to high yields. A few of them exhibit moderate cytotoxicity against various cancer cell lines such as HeLa (human epithelial cervical cancer), A549 (human lung carcinoma epithelial), DU145 (human prostate carcinoma epithelial) and MCF-7 (human breast adenocarcinoma).

Blockade of oncogenic IκB kinase activity in diffuse large B-cell lymphoma by bromodomain and extraterminal domain protein inhibitors.

In the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), NF-κB activity is essential for viability of the malignant cells and is sustained by constitutive activity of IκB kinase (IKK) in the cytoplasm. Here, we report an unexpected role for the bromodomain and extraterminal domain (BET) proteins BRD2 and BRD4 in maintaining oncogenic IKK activity in ABC DLBCL. IKK activity was reduced by small molecules targeting BET proteins as well as by genetic knockdown of BRD2 and BRD4 expression, thereby inhibiting downstream NF-κB-driven transcriptional programs and killing ABC DLBCL cells. Using a high-throughput platform to screen for drug-drug synergy, we observed that the BET inhibitor JQ1 combined favorably with multiple drugs targeting B-cell receptor signaling, one pathway that activates IKK in ABC DLBCL. The BTK kinase inhibitor ibrutinib, which is in clinical development for the treatment of ABC DLBCL, synergized strongly with BET inhibitors in killing ABC DLBCL cells in vitro and in a xenograft mouse model. These findings provide a mechanistic basis for the clinical development of BET protein inhibitors in ABC DLBCL, particularly in combination with other modulators of oncogenic IKK signaling.

Dopaminergic neurotoxicity of S-ethyl N,N-dipropylthiocarbamate (EPTC), molinate, and S-methyl-N,N-diethylthiocarbamate (MeDETC) in Caenorhabditis elegans.

Epidemiological studies corroborate a correlation between pesticide use and Parkinson's disease (PD). Thiocarbamate and dithiocarbamate pesticides are widely used and produce neurotoxicity in the peripheral nervous system. Recent evidence from rodent studies suggests that these compounds also cause dopaminergic (DAergic) dysfunction and altered protein processing, two hallmarks of PD. However, DAergic neurotoxicity has yet to be documented. We assessed DAergic dysfunction in Caenorhabditis elegans (C. elegans) to investigate the ability of thiocarbamate pesticides to induce DAergic neurodegeneration. Acute treatment with either S-ethyl N,N-dipropylthiocarbamate (EPTC), molinate, or a common reactive intermediate of dithiocarbamate and thiocarbamate metabolism, S-methyl-N,N-diethylthiocarbamate (MeDETC), to gradual loss of DAergic cell morphology and structure over the course of 6 days in worms expressing green fluorescent protein (GFP) under a DAergic cell specific promoter. HPLC analysis revealed decreased DA content in the worms immediately following exposure to MeDETC, EPTC, and molinate. In addition, worms treated with the three test compounds showed a drastic loss of DAergic-dependent behavior over a time course similar to changes in DAergic cell morphology. Alterations in the DAergic system were specific, as loss of cell structure and neurotransmitter content was not observed in cholinergic, glutamatergic, or GABAergic systems. Overall, our data suggest that thiocarbamate pesticides promote neurodegeneration and DAergic cell dysfunction in C. elegans, and may be an environmental risk factor for PD.