Preclinical Evidence Supporting Early Initiation of Citalopram Treatment in Machado-Joseph Disease

Spinocerebellar ataxias are dominantly inherited neurodegenerative disorders with no disease-modifying treatment. We previously identified the selective serotonin reuptake inhibitor citalopram as a safe and effective drug to be repurposed for Machado-Joseph disease. Pre-symptomatic treatment of transgenic (CMVMJD135) mice strikingly ameliorated mutant ataxin-3 (ATXN3) pathogenesis. Here, we asked whether citalopram treatment initiated at a post-symptomatic age would still show efficacy. We used a cohort of CMVMJD135 mice that shows increased phenotypic severity and faster disease progression (CMVMJD135hi) compared to the mice used in the first trial. Groups of hemizygous CMVMJD135hi mice were orally treated with citalopram. Behavior, protein analysis, and pathology assessment were performed blindly to treatment. Our results show that even when initiated after symptom onset, treatment of CMVMJD135hi mice with citalopram ameliorated motor coordination and balance, attenuating disease progression, albeit to a lesser extent than that seen with pre-symptomatic treatment initiation. There was no impact on ATXN3 aggregation, which contrasts with the robust reduction in ATXN3-positive inclusions observed in CMVMJD135 mice, when treated pre-symptomatically. Post-symptomatic treatment of CMVMJD135hi mice revealed, however, a limited neuroprotective effect by showing a tendency to repair cerebellar calbindin staining, and to increase the number of motor neurons and of NeuN-positive cells in certain brain regions. While supporting that early initiation of treatment with citalopram leads to a marked increase in efficacy, these results strengthen our previous observation that modulation of serotonergic signaling by citalopram is a promising therapeutic approach for Machado-Joseph disease even after symptom onset.European Regional Development Funds (FEDER), through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038. This article has been developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the FEDER. This work was also supported by FCT and COMPETE through the projects [PTDC/SAU-GMG/112617/2009] (to PM) and [EXPL/BIM-MEC/0239/2012] (to AT-C), by FCT through the project [POCI-01-0145-FEDER-016818 (PTDC/NEU-NMC/3648/2014)] (to PM), by National Ataxia foundation (to PM and to AT-C), and by Ataxia UK (to PM). SE, SD-S, SO, and AT-C were supported by the FCT individual fellowships, SFRH/BD/78554/2011, SFRH/BD/78388/2011, PD/BD/127818/2016, and SFRH/BPD/102317/2014, respectively. FCT fellowships are co-financed by POPH, QREN, Governo da República Portuguesa, and EU/FSEinfo:eu-repo/semantics/publishedVersio

Antidepressant stimulation of CDP-diacylglycerol synthesis does not require monoamine reuptake inhibition

<p>Abstract</p> <p>Background</p> <p>Recent studies demonstrate that diverse antidepressant agents increase the cellular production of the nucleolipid CDP-diacylglycerol and its synthetic derivative, phosphatidylinositol, in depression-relevant brain regions. Pharmacological blockade of downstream phosphatidylinositide signaling disrupted the behavioral antidepressant effects in rats. However, the nucleolipid responses were resistant to inhibition by serotonin receptor antagonists, even though antidepressant-facilitated inositol phosphate accumulation was blocked. Could the neurochemical effects be additional to the known effects of the drugs on monoamine transmitter transporters? To examine this question, we tested selected agents in serotonin-depleted brain tissues, in PC12 cells devoid of serotonin transporters, and on the enzymatic activity of brain CDP-diacylglycerol synthase - the enzyme that catalyzes the physiological synthesis of CDP-diacylglycerol.</p> <p>Results</p> <p>Imipramine, paroxetine, and maprotiline concentration-dependently increased the levels of CDP-diacylglycerol and phosphatidylinositides in PC12 cells. Rat forebrain tissues depleted of serotonin by pretreatment with <it>p</it>-chlorophenylalanine showed responses to imipramine or maprotiline that were comparable to respective responses from saline-injected controls. With fluoxetine, nucleolipid responses in the serotonin-depleted cortex or hippocampus were significantly reduced, but not abolished. Each drug significantly increased the enzymatic activity of CDP-diacylglycerol synthase following incubations with cortical or hippocampal brain tissues.</p> <p>Conclusion</p> <p>Antidepressants probably induce the activity of CDP-diacylglycerol synthase leading to increased production of CDP-diacylglycerol and facilitation of downstream phosphatidylinositol synthesis. Phosphatidylinositol-dependent signaling cascades exert diverse salutary effects in neural cells, including facilitation of BDNF signaling and neurogenesis. Hence, the present findings should strengthen the notion that modulation of brain phosphatidylinositide signaling probably contributes to the molecular mechanism of diverse antidepressant medications.</p

Scaffold dependent histone deacetylase (HDAC) inhibitor induced re-equilibration of the subcellular localization and post-translational modification state of class I HDACs

The mechanism of action of histone deacetylase inhibitors (HDACi) is mainly attributed to the inhibition of the deacetylase catalytic activity for their histone substrates. In this study, we analyzed the abundance of class I HDACs in the cytosolic, nuclear soluble and chromatin bound cellular fractions in breast cancer cells after HDACi treatment. We found that potent N-hydroxy propenamide-based HDACi induced a concentration dependent decrease in the HDAC1 associated with chromatin and a lasting concomitant increase in cytoplasmic HDAC1 while maintaining total protein expression. No such change occurred with HDAC2 or 8, however, an increase in cytoplasmic non-phosphorylated HDAC3 was also observed. The subcellular re-equilibration of HDAC1 was subsequent to the accumulation of acetylated histones and might be cell cycle dependent. This study suggests that the biological activity of a subset of N-hydroxy propenamide-based HDACi may stem from direct competition with histone substrates of HDACs as well as from spatial separation from their substrates in the nucleus and/or change in post-translational modification status of HDACs

Synergy Trap for Guardian Angels of DNA: Unraveling the Anticancer Potential of Phthalazinone -Thiosemicarbazone Hybrids through Dual PARP-1 and TOPO-I Inhibition

Targeting DNA repair, like PARP-1 and TOPO-I, shows promise in cancer therapy. However, resistance to single agents requires complex and costly combination strategies with significant side effects. Thus, there's an urgent need for single agents with dual inhibition. Current dual inhibitors focusing on the C-4 position of the phthalazinone core for PARP inhibition often have high molecular weights. Clinical use of PARP inhibitors is limited by hematological and other toxicities from concurrent PARP-2 inhibition. They're mainly effective in gynecological cancers, despite high PARP-1 and TOPO-I expression in various cancers. Moreover, their efficacy is limited to BRCA1-expressing breast cancer. In this study, we synthesized 27 dual inhibitors for PARP-1 and TOPO-I with molecular weights below 500 g/mol through hybridizing a phthalazinone core with a thiosemicarbazone linker. Among these, 6c demonstrated exceptional broad spectrum and potency against the NCI 60 cancer cell lines, with GI50 values from 1.65 to 5.63 µM. Notably, 6c exposed the highest PARP-1 inhibition (IC50 = 32.2 ± 3.26 nM) and a selectivity over PARP-2 (IC50 = 2844 ± 111 nM). Furthermore, 6c's inhibition of TOPO-I (IC50 = 46.2 ± 3.3 nM) surpassed the control camptothecin by eleven-fold. Mechanistically, 6c disrupted the cell cycle at the S phase, induced apoptosis, and displayed a favorable safety profile against normal cells. Compound 6c induced PARP trapping and synthetic lethality and showed high efficacy on BRCA1- expressing cell lines. So, decreasing the likelihood of cancer cell resistance to chemotherapy. Drug-likeness predictions and molecular modeling were also performed

In Vitro Characterization of Inhibitors for Lung A549 and Leukemia K562 Cell Lines from Fungal Transformation of Arecoline Supported by In Silico Docking to M3-mAChR and ADME Prediction

The search for anticancer drugs is of continuous interest. Arecoline is an alkaloid with anticancer activity. Herein, the metabolism of arecoline through fungal transformation was investigated for the discovery of potential anticancer drugs with higher activity and selectivity. Compounds 1&ndash;5 were isolated, and their structures were fully elucidated using various spectroscopic analyses, including 1D and 2D NMR, ESIMS, and HRESIMS. This is the first report for the isolation of compounds 1 and 2. An MTT assay was performed to determine the cytotoxic activity of arecoline and its metabolites in vitro using non-small-cell lung cancer A549 and leukemia K562 cell lines compared to staurosporine and doxorubicin as positive controls. For the non-small-cell lung A549 cell line, arecoline hydrobromide, staurosporine, and doxorubicin resulted in IC50 values of 11.73 &plusmn; 0.71 &micro;M, 10.47 &plusmn; 0.64 &micro;M, and 5.05 &plusmn; 0.13 &micro;M, respectively, while compounds 1, 3, and 5 exhibited IC50 values of 3.08 &plusmn; 0.19 &micro;M, 7.33 &plusmn; 0.45 &micro;M, and 3.29 &plusmn; 0.20 &micro;M, respectively. For the leukemia K562 cell line, the IC50 values of arecoline hydrobromide, staurosporine, and doxorubicin were 15.3 &plusmn; 1.08 &micro;M, 5.07 &plusmn; 0.36 &micro;M, and 6.94 &plusmn; 0.21 &micro;M, respectively, while the IC50 values of compounds 1, 3 and 5 were 1.56 &plusmn; 0.11 &micro;M, 3.33 &plusmn; 0.24 &micro;M, and 2.15 &plusmn; 0.15 &micro;M, respectively. The selectivity index value of these compounds was higher than 3. These results indicated that compounds 1, 3, and 5 are very strong cytotoxic agents with higher activity than the positive controls and good selectivity toward the tested cancer cell lines. Cell cycle arrest was then studied by flow cytometry to investigate the apoptotic mechanism. Docking simulation revealed that most compounds possessed good binding poses and favorable protein-ligand interactions with muscarinic acetylcholine receptor M3-mAChR protein. In silico study of pharmacokinetics using SwissADME predicted compounds 1&ndash;5 to be drug-like with a high probability of good oral bioavailability

Terminators or Guardians? Design, Synthesis, and Cytotoxicity Profiling of Chalcone-Sulfonamide Hybrids

With a “less is more” philosophy, a series of 15 chalcone-sulfonamide hybrids were designed anticipating synergistic anticancer activity. The aromatic sulfonamide moiety was included as a known direct inhibitor of carbonic anhydrase IX activity through its zinc chelating property. The chalcone moiety was incorporated as an electrophilic stressor to indirectly inhibit carbonic anhydrase IX cellular activity. Screening by the Developmental Therapeutics Program of the National Cancer Institute, NCI-60, revealed that 12 derivatives were potent inhibitors of cancer cell growth in multiple cell lines and were promoted to the five-dose screen. The cancer cell growth inhibition profile indicated sub- to two-digit micromolar potency (GI50 down to 0.3 μM and LC50 as low as 4 μM) against colorectal carcinoma cells, in particular. Unexpectedly, most compounds demonstrated low to moderate potency as direct inhibitors of carbonic anhydrase catalytic activity in vitro, with 4d being the most potent, having an average Ki value of 4 μM. Compound 4j showed ca. six-fold selectivity to carbonic anhydrase IX over the other tested isoforms in vitro. Cytotoxicity of both 4d and 4j in live HCT116, U251, and LOX IMVI cells under hypoxic conditions confirmed their targeting of carbonic anhydrase activity. Elevation of oxidative cellular stress was stipulated from the increase in Nrf2 and ROS levels in 4j-treated colorectal carcinoma, HCT116, cells compared to the control. Compound 4j arrested the cell cycle of HCT116 cells at the G1/S phase. In addition, both 4d and 4j showed up to 50-fold cancer cell selectivity compared to the non-cancerous HEK293T cells. Accordingly, this study presents 4d and 4j being new, synthetically accessible, simplistically designed derivatives as potential candidates to be further developed as anticancer therapeutics

Isonicotinic acid <i>N</i>-oxide, from isoniazid biotransformation by <i>Aspergillus niger</i>, as an InhA inhibitor antituberculous agent against multiple and extensively resistant strains supported by <i>in silico</i> docking and ADME prediction

Biotransformation of isoniazid produced isonicotinic acid (1), isonicotinic acid N-oxide (2), and isonicotinamide (3) which were isolated by column chromatography using silica gel and Sephadex LH 20 and elucidated using various spectroscopies. This is the first report for isolation of 2. Antituberculosis activity was evaluated against Mycobacterium tuberculosis strains: drug sensitive (DS), multiple drug resistant (MDR) and extensively drug resistant (XDR). 1-3 and isoniazid showed MICs of 63.49, 0.22, 15.98 and 0.88 µM, respectively, against the DS strain. For the MDR strain, 2 and 3 exhibited MICs of 28.06 and > 1000 µM, respectively, while 1 was inactive. Moreover, 2 had an MIC of 56.19 µM against XDR strain, while 1 and 3 were inactive. Docking simulation using enoyl ACP reductase (InhA) revealed favorable protein-ligand interactions. In silico study of pharmacokinetics and hepatotoxicity predicted 1-3 to have good oral bioavailability and 2 to have a lower hepatoxicity probability than isoniazid.</p