Glucose-6-phosphate dehydrogenase plays a central role in the response of tomato (Solanum lycopersicum) plants to short and long-term drought

The present study was undertaken to investigate the expression, occurrence and activity of glucose 6 phosphate dehydrogenase (G6PDH e EC 1.1.1.49), the key-enzyme of the Oxidative Pentose Phosphate Pathway (OPPP), in tomato plants (Solanum lycopersicum cv. Red Setter) exposed to short- and long-term drought stress. For the first time, drought effects have been evaluated in plants under different growth conditions: in hydroponic laboratory system, and in greenhouse pots under controlled conditions; and in open field, in order to evaluate drought response in a representative agricultural environment. Interestingly, changes observed appear strictly associated to the induction of well known stress response mechanisms, such as the increase of proline synthesis, accumulation of chaperone Hsp70, and ascorbate peroxidase. Results show significant increase in total activity of G6PDH, and specifically in expression and occurrence of cytosolic isoform (cy-G6PDH) in plants grown in any cultivation system upon drought. Intriguingly, the results clearly suggest that abscissic acid (ABA) pathway and signaling cascade (protein phosphatase 2C e PP2C) could be strictly related to increased G6PDH expression, occurrence and activities. We hypothesized for G6PDH a specific role as one of the main reductants’ suppliers to counteract the effects of drought stress, in the light of converging evidences given by young and adult tomato plants under stress of different duration and intensity

COMBINING TRANSLATION READTHROUGH INDUCING DRUGS AND NONSENSE MEDIATED DECAY PATWHAY INHIBITION TO THE CFTR RESCUE IN CYSTIC FIBROSIS CELL MODEL SYSTEM

Nonsense mutations affect 10% of patients with cystic fibrosis and produce a premature termination codon in CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) mRNA causing early termination of translation and leading to lack of CFTR function. A potential therapy for nonsense mutations provides the use of small molecules able to overcome the premature stop codon (PTC) by a readthrough mechanism that lead to synthesis a complete CFTR protein. Despite the good results obtained from this approach, TRIDs efficiency is considerably reduced by the poor amount of target transcript, that is the mRNA containing the PTC. The readthrough, indeed, does not occur on the totality of target transcripts because of their degradation due to the nonsense mediated decay pathway (NMD). This pathway provides the degradation of mRNA harboring premature stop codon to prevent the production of altered polypeptides. In contrast, the activity of this pathway interferes with the effectiveness of the readthrough drugs, limiting the mRNA concentration of the target protein. Thus, a promising strategy for nonsense mutation treatment is a combined use of readthrough agents and factors that attenuate the nonsense mRNA decay. By silencing the UPF1 mRNA/protein, the activity of the NMD pathway was reduced, in FRT cells CFTR W1282X. Alternatively, caffeine was used as specific inhibitor of the UPF1 activity, to increase the efficiency of readthrough molecules (NV848 and NV914) in FRT cells CFTRW1282X cells. In both cases, the combined treatment: NV848 or NV914/caffeine and NV848 or NV914/UPF1siRNA caused an increase of CFTRW1282X mRNA level followed by the rescue of the CFTR expression and functionality. However, unexpectedly, despite the higher CFTRW1282X mRNA level in caffeine treated samples, both expression and functionality CFTR rescue resulted slightly lower than the recovery achieved by UPF1 silencing. Our results indicate that modulation of NMD pathway, although still to be optimized, could be a promising approach in order to increase TRIDs effects in presence of stop mutations

The chemistry and pharmacology of citrus limonoids

Citrus limonoids (CLs) are a group of highly oxygenated terpenoid secondary metabolites found mostly in the seeds, fruits and peel tissues of citrus fruits such as lemons, limes, oranges, pumellos, grapefruits, bergamots, and mandarins. Represented by limonin, the aglycones and glycosides of CLs have shown to display numerous pharmacological activities including anticancer, antimicrobial, antioxidant, antidiabetic and insecticidal among others. In this review, the chemistry and pharmacology of CLs are systematically scrutinised through the use of medicinal chemistry tools and structure-activity relationship approach. Synthetic derivatives and other structurally-related limonoids from other sources are include in the analysis. With the focus on literature in the past decade, the chemical classification of CLs, their physico-chemical properties as drugs, their biosynthesis and enzymatic modifications, possible ways of enhancing their biological activities through structural modifications, their ligand efficiency metrics and systematic graphical radar plot analysis to assess their developability as drugs are among those discussed in detai

Inhibition of voltage-gated sodium channels by sumatriptan bioisosteres

Voltage-gated sodium channels are known to play a pivotal role in perception and transmission of pain sensations. Gain-of-function mutations in the genes encoding the peripheral neuronal sodium channels, hNav1.7-1.9, cause human painful diseases. Thus while treatment of chronic pain remains an unmet clinical need, sodium channel blockers are considered as promising druggable targets. In a previous study, we evaluated the analgesic activity of sumatriptan, an agonist of serotonin 5HT1B/D receptors, and some new chiral bioisosteres, using the hot plate test in the mouse. Interestingly, we observed that the analgesic effectiveness was not necessarily correlated to serotonin agonism. In this study, we evaluated whether sumatriptan and its congeners may inhibit heterologously expressed hNav1.7 sodium channels using the patch-clamp method. We show that sumatriptan blocks hNav1.7 channels only at very high, supratherapeutic concentrations. In contrast, its three analogs, namely 20b, (R)-31b, and (S)-22b, exert a dose and use-dependent sodium channel block. At 0.1 and 10 Hz stimulation frequencies, the most potent compound, (S)-22b, was 4.4 and 1.7 fold more potent than the well-known sodium channel blocker mexiletine. The compound induces a negative shift of voltage dependence of fast inactivation, suggesting higher affinity to the inactivated channel. Accordingly, we show that (S)-22b likely binds the conserved local anesthetic receptor within voltage-gated sodium channels. Combining these results with the previous ones, we hypothesize that use-dependent sodium channel blockade contributes to the analgesic activity of (R)-31b and (S)-22b. These later compounds represent promising lead compounds for the development of efficient analgesics, the mechanism of action of which may include a dual action on sodium channels and 5HT1D receptors

Molecular Insights into the Local Anesthetic Receptor within Voltage-Gated Sodium Channels Using Hydroxylated Analogs of Mexiletine

We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, directly blocked voltage-gated sodium channels, whereas salbutamol and nadolol did not (Desaphy et al., 2003), suggesting the presence of two hydroxyl groups on the aromatic moiety of the drugs as a molecular requisite for impeding sodium channel block. To verify such an hypothesis, we synthesized five new mexiletine analogs by adding one or two hydroxyl groups to the aryloxy moiety of the sodium channel blocker and tested these compounds on hNav1.4 channels expressed in HEK293 cells. Concentration–response relationships were constructed using 25-ms-long depolarizing pulses at −30 mV applied from an holding potential of −120 mV at 0.1 Hz (tonic block) and 10 Hz (use-dependent block) stimulation frequencies. The half-maximum inhibitory concentrations (IC50) were linearly correlated to drug lipophilicity: the less lipophilic the drug, minor was the block. The same compounds were also tested on F1586C and Y1593C hNav1.4 channel mutants, to gain further information on the molecular interactions of mexiletine with its receptor within the sodium channel pore. In particular, replacement of Phe1586 and Tyr1593 by non-aromatic cysteine residues may help in the understanding of the role of π–π or π–cation interactions in mexiletine binding. Alteration of tonic block suggests that the aryloxy moiety of mexiletine may interact either directly or indirectly with Phe1586 in the closed sodium channel to produce low-affinity binding block, and that this interaction depends on the electrostatic potential of the drug aromatic tail. Alteration of use-dependent block suggests that addition of hydroxyl groups to the aryloxy moiety may modify high-affinity binding of the drug amine terminal to Phe1586 through cooperativity between the two pharmacophores, this effect being mainly related to drug lipophilicity. Mutation of Tyr1593 further impaired such cooperativity. In conclusion, these results confirm our former hypothesis by showing that the presence of hydroxyl groups to the aryloxy moiety of mexiletine greatly reduced sodium channel block, and provide molecular insights into the intimate interaction of local anesthetics with their receptor

Microwave-Assisted Extraction of Bioactive Compounds from Lentil Wastes: Antioxidant Activity Evaluation and Metabolomic Characterization

The recovery of industrial by-products is part of the zero-waste circular economy. Lentil seed coats are generally considered to be a waste by-product. However, this low-value by-product is rich in bioactive compounds and may be considered an eco-friendly source of health-promoting phytochemicals. For the first time, a sustainable microwave-assisted extraction technique was applied, and a solvent screening was carried out to enhance the bioactive compound content and the antioxidant activity of green and red lentil hull extracts. With respect to green lentil hull extracts that were obtained with different solvents, the aqueous extract of the red lentil seed coats showed the highest total phenolic and total flavonoid content (TPC = 28.3 ± 0.1 mg GAE/g dry weight, TFC = 1.89 ± 0.01 mg CE/100 mg dry weight, respectively), as well as the highest antioxidant activity, both in terms of the free radical scavenging activity (ABTS, 39.06 ± 0.73 mg TE/g dry weight; DPPH, IC50 = 0.39 μg/mL) and the protection of the neuroblastoma cell line (SH-SY5Y, IC50 = 10.1 ± 0.6 μg/mL), the latter of which has never been investigated so far. Furthermore, a metabolite discovery analysis was for the first time performed on the aqueous extracts of both cultivars using an HPLC separation which was coupled with an Orbitrap-based high-Resolution Mass Spectrometry technique

Hunting for lubeluzole analogues as antimyotonic agents with reduced cardiac liability

Lubeluzole is a neuroprotective agent displaying antimyotonic activity. Lubeluzole clinical development as an antiischemic drug was discontinued due to a lack of efficacy in human trials and possible cardiac toxicity. Since lubeluzole is a potent inhibitor of the hERG channel, involved in long QT syndromes and the potentially fatal cardiac arrhythmia Torsade de Pointes, a series of lubeluzole analogues were prepared to investigate the structural requirements to reduce the affinity for hERG channels to possibly obtain safe antimyotonic drugs. Compound 16o was identified as the less potent hERG blocker possibly endowed with lower cardiac liability in comparison with the parent compound. Antimyotonic activity of 16o was also investigated in vitro on hNav1.4 and higher use-dependence was observed in comparison to lubeluzole, thus suggesting greater selectivity toward highly excited tissues, such as the myotonic muscle. To further verify the cardiac safety of 16o, patch-clamp experiments on hNav1.5 were also carried out and a 3-fold reduction of potency in comparison with hNav1.4 in phasic block was observed. In vivo evaluation of the antimyotonic activity showed unintended effects on rat motor performance. Ex vivo studies suggested calcium channel blocking activity as a possible off-target source of the 16o unintended effects, also reinforced by possible interaction with β2 receptors, as indicated by in vitro binding assays and in silico studies. In conclusion, we think our results may support the rational design of lubeluzole analogues endowed with both antimyotonic activity and lower hERG liability

Synthesis and Evaluation of Voltage-Gated Sodium Channel Blocking Pyrroline Derivatives Endowed with Both Antiarrhythmic and Antioxidant Activities

Under the hypothesis that cardioprotective agents might benefit from synergism between antiarrhythmic activity and antioxidant properties, a small series of mexiletine analogues were coupled with the 2,2,5,5-tetramethylpyrroline moiety, known for its antioxidant effect, in order to obtain dual-acting drugs potentially useful in the protection of the heart against post-ischemic reperfusion injury. The pyrroline derivatives reported herein were found to be more potent as antiarrhythmic agents than mexiletine and displayed antioxidant activity. The most interesting tetramethylpyrroline congener, a tert-butyl-substituted analogue, was at least 100 times more active as an antiarrhythmic than mexiletine

C1Q+ TPP1+ macrophages promote colon cancer progression through SETD8-driven p53 methylation

Background: In many tumors, the tumor suppressor TP53 is not mutated, but functionally inactivated. However, mechanisms underlying p53 functional inactivation remain poorly understood. SETD8 is the sole enzyme known to mono-methylate p53 on lysine 382 (p53K382me1), resulting in the inhibition of its pro-apoptotic and growth-arresting functions. Methods: We analyzed SETD8 and p53K382me1 expression in clinical colorectal cancer (CRC) and inflammatory bowel disease (IBD) samples. Histopathological examinations, RNA sequencing, ChIP assay and preclinical in vivo CRC models, were used to assess the functional role of p53 inactivation in tumor cells and immune cell infiltration. Results: By integrating bulk RNAseq and scRNAseq approaches in CRC patients, SETD8-mediated p53 regulation resulted the most significantly enriched pathway. p53K382me1 expression was confined to colorectal cancer stem cells (CR-CSCs) and C1Q+ TPP1+ tumor-associated macrophages (TAMs) in CRC patient tissues, with high levels predicting decreased survival probability. TAMs promote p53 functional inactivation in CR-CSCs through IL-6 and MCP-1 secretion and increased levels of CEBPD, which directly binds SETD8 promoter thus enhancing its transcription. The direct binding of C1Q present on macrophages and C1Q receptor (C1QR) present on cancer stem cells mediates the cross-talk between the two cell compartments. As monotherapy, SETD8 genetic and pharmacological (UNC0379) inhibition affects the tumor growth and metastasis formation in CRC mouse avatars, with enhanced effects observed when combined with IL-6 receptor targeting. Conclusions: These findings suggest that p53K382me1 may be an early step in tumor initiation, especially in inflammation-induced CRC, and could serve as a functional biomarker and therapeutic target in adjuvant setting for advanced CRCs

Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour

Previous efforts to control cellular behaviour have largely relied upon various forms of genetic engineering. Once the genetic content of a living cell is modified, the behaviour of that cell typically changes as well. However, other methods of cellular control are possible. All cells sense and respond to their environment. Therefore, artificial, non-living cellular mimics could be engineered to activate or repress already existing natural sensory pathways of living cells through chemical communication. Here we describe the construction of such a system. The artificial cells expand the senses of Escherichia coli by translating a chemical message that E. coli cannot sense on its own to a molecule that activates a natural cellular response. This methodology could open new opportunities in engineering cellular behaviour without exploiting genetically modified organisms