ß-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway.

ß-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that ß-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of ß-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. ß-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

Biomimetic noncationic lipid nanoparticles for mRNA delivery.

Although the tremendous progress has been made for mRNA delivery based on classical cationic carriers, the excess cationic charge density of lipids was necessary to compress mRNA through electrostatic interaction, and with it comes inevitably adverse events including the highly inflammatory and cytotoxic effects. How to develop the disruptive technologies to overcome cationic nature of lipids remains a major challenge for safe and efficient mRNA delivery. Here, we prepared noncationic thiourea lipids nanoparticles (NC-TNP) to compress mRNA by strong hydrogen bonds interaction between thiourea groups of NC-TNP and the phosphate groups of mRNA, abandoning the hidebound and traditional electrostatic force to construct mRNA-cationic lipids formulation. NC-TNP was a delivery system for mRNA with simple, convenient, and repeatable preparation technology and showed negligible inflammatory and cytotoxicity side effects. Furthermore, we found that NC-TNP could escape the recycling pathway to inhibit the egress of internalized nanoparticles from the intracellular compartment to the extracellular milieu which was a common fact in mRNA-LNP (lipid nanoparticles) formulation. Therefore, NC-TNP-encapsulated mRNA showed higher gene transfection efficiency in vitro and in vivo than mRNA-LNP formulation. Unexpectedly, NC-TNP showed spleen targeting delivery ability with higher accumulation ratio (spleen/liver), compared with traditional LNP. Spleen-targeting NC-TNP with mRNA exhibited high mRNA-encoded antigen expression in spleen and elicited robust immune responses. Overall, our work establishes a proof of concept for the construction of a noncationic system for mRNA delivery with good inflammatory safety profiles, high gene transfection efficiency, and spleen-targeting delivery to induce permanent and robust humoral and cell-mediated immunity for disease treatments.

Antioxidants are ineffective at quenching reactive oxygen species inside bacteria and should not be used to diagnose oxidative stress.

A wide variety of stresses have been proposed to exert killing effects upon bacteria by stimulating the intracellular formation of reactive oxygen species (ROS). A key part of the supporting evidence has often been the ability of antioxidant compounds to protect the cells. In this study, some of the most-used antioxidants-thiourea, glutathione, N-acetylcysteine, and ascorbate-have been examined. Their ability to quench superoxide and hydrogen peroxide was verified in vitro, but the rate constants were orders of magnitude too slow for them to have an impact upon superoxide and peroxide concentrations in vivo, where these species are already scavenged by highly active enzymes. Indeed, the antioxidants were unable to protect the growth and ROS-sensitive enzymes of E. coli strains experiencing authentic oxidative stress. Similar logic posits that antioxidants cannot substantially quench hydroxyl radicals inside cells, which contain abundant biomolecules that react with them at diffusion-limited rates. Indeed, antioxidants were able to protect cells from DNA damage only if they were applied at concentrations that slow metabolism and growth. This protective effect was apparent even under anoxic conditions, when ROS could not possibly be involved, and it was replicated when growth was similarly slowed by other means. Experimenters should discard the use of antioxidants as a way of detecting intracellular oxidative stress and should revisit conclusions that have been based upon such experiments. The notable exception is that these compounds can effectively degrade hydrogen peroxide from environmental sources before it enters cells.

A chemical timer approach to delayed chemiluminescence.

Although the onset time of chemical reactions can be manipulated by mechanical, electrical, and optical methods, its chemical control remains highly challenging. Herein, we report a chemical timer approach for manipulating the emission onset time of chemiluminescence (CL) reactions. A mixture of Mn2+, NaHCO3, and a luminol analog with H2O2 produced reactive oxygen species (ROS) radicals and other superoxo species (superoxide containing complex) with high efficiency, accompanied by strong and immediate CL emission. Surprisingly, the addition of thiourea postponed CL emission in a concentration-dependent manner. The delay was attributed to a slow-generation-scavenging mechanism, which was found to be generally applicable not only to various types of CL reagents and ROS radical scavengers but also to popular chromogenic reactions. The precise regulation of CL kinetics was further utilized in dynamic chemical coding with improved coding density and security. This approach provides a powerful platform for engineering chemical reaction kinetics using chemical timers, which is of application potential in bioassays, biosensors, CL microscopic imaging, microchips, array chips, and informatics.

Functional asymmetry of the urea transporter UT-B in human red blood cells.

We studied urea, thiourea, and methylurea transport and interaction in human red blood cells (RBCs) under conditions of self-exchange (SE), net efflux (NE), and net influx (NI) at pH 7.2. We combined four methods, a four-centrifuge technique, the Millipore-Swinnex filtering technique, the continuous flow tube method, and a continuous pump method to measure the transport of the 14C-labeled compounds. Under SE conditions, both urea and thiourea show perfect Michaelis-Menten kinetics with half-saturation constants, K½,SE (mM), of ≈300 (urea) and ≈20 (thiourea). The solutes show no concentration-dependent saturation under NE conditions. Under NI conditions, transport displays saturation or self-inhibition kinetics with a K½,NI (mM) of ≈210 (urea) and ≈20 (thiourea). Urea, thiourea, and methylurea are competitive inhibitors of the transport of analog solutes. This study supports the hypothesis that the three compounds share the same urea transport system (UT-B). UT-B functions asymmetrically as it saturates from the outside only under SE and NI conditions, whereas it functions as a high-capacity channel-like transporter under NE conditions. When the red blood cell enters the urea-rich kidney tissue, self-inhibition reduces the urea uptake in the cell. When the cell leaves the kidney, the channel-like function of UT-B implies that intracellular urea rapidly equilibrates with external urea. The net result is that the cell during the passage in the kidney capillaries carries urea to the kidney to be excreted while the urea transfer from the kidney via the bloodstream is minimized.NEW & NOTEWORTHY The kinetics of urea transport in red blood cells was determined by means of a combination of four methods that ensures a high time resolution. In the present study, we disclose that the urea transporter UT-B functions highly asymmetric being channel-like with no saturation under conditions of net efflux and saturable under conditions of net influx and self-exchange in the concentration range 1-1,000 mM (pH 7.2 and 25-38 °C).

Endoplasmic reticulum stress-induced senescence in human lung fibroblasts.

Loss of proteostasis and cellular senescence have been previously established as characteristics of aging; however, their interaction in the context of lung aging and potential contributions to aging-associated lung remodeling remains understudied. In this study, we aimed to characterize endoplasmic reticulum (ER) stress response, cellular senescence, and their interaction in relation to extracellular matrix (ECM) production in lung fibroblasts from young (25-45 yr) and old (>60 yr) humans. Fibroblasts from young and old patients without significant preexisting lung disease were exposed to vehicle, MG132, etoposide, or salubrinal. Afterward, cells and cell lysates or supernatants were analyzed for ER stress, cellular senescence, and ECM changes using protein analysis, proliferation assay, and senescence-associated beta-galactosidase (SA-ß-Gal) staining. At baseline, fibroblasts from aging individuals showed increased levels of ER stress (ATF6 and PERK), senescence (p21 and McL-1), and ECM marker (COL1A1) compared to those from young individuals. Upon ER stress induction and etoposide exposure, fibroblasts showed an increase in senescence (SA-ß-Gal, p21, and Cav-1), ER stress (PERK), and ECM markers (COL1A1 and LUM) compared to vehicle. Additionally, IL-6 and IL-8 levels were increased in the supernatants of MG132- and etoposide-treated fibroblasts, respectively. Finally, the ER stress inhibitor salubrinal decreased the expression of p21 compared to vehicle and MG132 treatments; however, salubrinal inhibited COL1A1 but not p21 expression in MG132-treated fibroblasts. Our study suggests that ER stress response plays an important role in establishment and maintenance of a senescence phenotype in lung fibroblasts and therefore contributes to altered remodeling in the aging lung.NEW & NOTEWORTHY The current study establishes functional links between endoplasmic reticulum (ER) stress and cellular senescence per se in the specific context of aging human lung fibroblasts. Recognizing that the process of aging per se is complex, modulated by the myriad of lifelong and environmental exposures, it is striking to note that chronic ER stress may play a crucial role in the establishment and maintenance of cellular senescence in lung fibroblasts.

One-to-Nine Single Spectroscopic Intelligent Probe for Risk Assessment of Multiple Metals in Drinking Water.

26% of the world's population lacks access to clean drinking water; clean water and sanitation are major global challenges highlighted by the UN Sustainable Development Goals, indicating water security in public water systems is at stake today. Water monitoring using precise instruments by skilled operators is one of the most promising solutions. Despite decades of research, the professionalism-convenience trade-off when monitoring ubiquitous metal ions remains the major challenge for public water safety. Thus, to overcome these disadvantages, an easy-to-use and highly sensitive visual method is desirable. Herein, an innovative strategy for one-to-nine metal detection is proposed, in which a novel thiourea spectroscopic probe with high 9-metal affinity is synthesized, acting as "one", and is detected based on the 9 metal-thiourea complexes within portable spectrometers in the public water field; this is accomplished by nonspecialized personnel as is also required. During the processing of multimetal analysis, issues arise due to signal overlap and reproducibility problems, leading to constrained sensitivity. In this innovative endeavor, machine learning (ML) algorithms were employed to extract key features from the composite spectral signature, addressing multipeak overlap, and completing the detection within 30-300 s, thus achieving a detection limit of 0.01 mg/L and meeting established conventional water quality standards. This method provides a convenient approach for public drinking water safety testing.

Promoting the Anticancer Activity with Multidentate Furan-2-Carboxamide Functionalized Aroyl Thiourea Chelation in Binuclear Half-Sandwich Ruthenium(II) Complexes.

A new set of binuclear arene ruthenium complexes [Ru2(p-cymene)2(k4-N2OS)(L1-L3)Cl2] (Ru2L1-Ru2L3) encompassing furan-2-carboxamide-based aroylthiourea derivatives (H2L1-H2L3) was synthesized and characterized by various spectral and analytical techniques. Single-crystal XRD analysis unveils the N^O and N^S mixed monobasic bidentate coordination of the ligands constructing N, S, Cl/N, O, and Cl legged piano stool octahedral geometry. DFT analysis demonstrates the predilection for the formation of stable arene ruthenium complexes. In vitro antiproliferative activity of the complexes was examined against human cervical (HeLa), breast (MCF-7), and lung (A549) cancerous and noncancerous monkey kidney epithelial (Vero) cells. All the complexes are more efficacious against HeLa and MCF-7 cells with low inhibitory doses (3.86-11.02 µM). Specifically, Ru2L3 incorporating p-cymene and -OCH3 fragments exhibits high lipophilicity, significant cytotoxicity against cancer cells, and lower toxicity on noncancerous cells. Staining analysis indicates the apoptosis-associated cell morphological changes expressively in MCF-7 cells. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) analyses reveal that Ru2L3 can raise ROS levels, reduce MMP, and trigger mitochondrial dysfunction-mediated apoptosis. The catalytic oxidation of glutathione (GSH) to its disulfide form (GSSG) by the complexes may simultaneously increase the ROS levels, alluding to their observed cytotoxicity and apoptosis induction. Flow cytometry determined the quantitative classification of late apoptosis and S-phase arrest in MCF-7 and HeLa cells. Western blotting analysis confirmed that the complexes promote apoptosis by upregulating Caspase-3 and Caspase-9 and downregulating BCL-2. Molecular docking studies unfolded the strong binding affinities of the complexes with VEGFR2, an angiogenic signaling receptor, and BCL2, Cyclin D1, and HER2 proteins typically overexpressed on tumor cells.

A novel analytical approach for the determination of ethylene-thiourea and propylene-thiourea in vegetal foodstuffs by high-performance liquid chromatography hyphenated to inductively coupled plasma-tandem mass spectrometry.

This study reports a novel analytical approach for the simultaneous determination of ethylene-thiourea (ETU) and propylene-thiourea (PTU) in fruits and vegetables by (reverse phase) high-performance liquid chromatography (HPLC) coupled to inductively coupled plasma-tandem mass spectrometry (ICP-QQQMS or ICP-MS/MS). A baseline separation of ETU and PTU was achieved in less than 5 min. A robust method validation by using the accuracy profile approach was performed by carrying out four measurement series in duplicate at six different levels over a timespan of 4 weeks (different days). The recovery factors ranged from 87 to 101% for ETU and from 98 to 99% for PTU (depending on the spiking level). The coefficient of variation in terms of repeatability (CVr) ranged from 1 to 4.7% for ETU and from 1.8 to 3.9% for PTU (depending also on the analyte level) while the coefficient of variation in terms of intermediate reproducibility (CVR) ranged from 3.4 to 10% for ETU and from 1.8 to 10.8% for PTU. The limit of quantification was 0.022 mg kg-1 (wet weight) for ETU and 0.010 mg kg-1 (ww) for PTU. This novel approach was proved to be highly robust and suitable for the determination of ETU and PTU in foodstuffs of vegetal origin.

Identification of (E)-1-((1H-indol-3-yl)methylene)-4-substitute-thiosemicarbazones as potential anti-hepatic fibrosis agents.

Liver fibrosis remains a global health challenge due to its rapidly rising prevalence and limited treatment options. The orphan nuclear receptor Nur77 has been implicated in regulation of autophagy and liver fibrosis. Targeting Nur77-mediated autophagic flux may thus be a new promising strategy against hepatic fibrosis. In this study, we synthesized four types of Nur77-based thiourea derivatives to determine their anti-hepatic fibrosis activity. Among the synthesized thiourea derivatives, 9e was the most potent inhibitor of hepatic stellate cells (HSCs) proliferation and activation. This compound could directly bind to Nur77 and inhibit TGF-ß1-induced α-SMA and COLA1 expression in a Nur77-dependent manner. In vivo, 9e significantly reduced CCl4-mediated hepatic inflammation response and extracellular matrix (ECM) production, revealing that 9e is capable of blocking the progression of hepatic fibrosis. Mechanistically, 9e induced Nur77 expression and enhanced autophagic flux by inhibiting the mTORC1 signaling pathway in vitro and in vivo. Thus, the Nur77-targeted lead 9e may serve as a promising candidate for treatment of chronic liver fibrosis.

Discovery of novel pyrazole based Urea/Thiourea derivatives as multiple targeting VEGFR-2, EGFRWT, EGFRT790M tyrosine kinases and COX-2 Inhibitors, with anti-cancer and anti-inflammatory activities.

A novel series of pyrazole derivatives with urea/thiourea scaffolds 16a-l as hybrid sorafenib/erlotinib/celecoxib analogs was designed, synthesized and tested for its VEGFR-2, EGFRWT, EGFRT790M tyrosine kinases and COX-2, pro-inflammatory cytokines TNF-α and IL-6 inhibitory activities. All the tested compounds showed excellent COX-2 selectivity index in range of 18.04-47.87 compared to celecoxib (S.I. = 26.17) and TNF-α and IL-6 inhibitory activities (IC50 = 5.0-7.50, 6.23-8.93 respectively, compared to celecoxib IC50 = 8.40 and 8.50, respectively). Screening was carried out against 60 human cancer cell lines by National Cancer Institute (NCI), compounds 16a, 16c, 16d and 16 g were the most potent inhibitors with GI% ranges of 100 %, 99.63-87.02 %, 98.98-43.10 % and 98.68-23.62 % respectively, and with GI50 values of 1.76-15.50 µM, 1.60-5.38 µM, 1.68-7.39 µM and 1.81-11.0 µM respectively, in addition, of showing good safety profile against normal cell line (F180). Moreover, compounds 16a, 16c, 16d and 16 g had cell cycle arrest at G2/M phase with induced necrotic percentage compared to sorafenib of 2.06 %, 2.47 %, 1.57 %, 0.88 % and 1.83 % respectively. Amusingly, compounds 16a, 16c, 16d and 16 g inhibited VEGFR-2 with IC50 of 25 nM, 52 nM, 324 nM and 110 nM respectively, compared to sorafenib (IC50 = 85 nM), and had excellent EGFRWT and EGFRT790M kinase inhibitory activities (IC50 = 94 nM, 128 nM, 160 nM, 297 nM), (10 nM, 25 nM, 36 nM and 48 nM) respectively, compared to both erlotinib and osimertinib (IC50 = 114 nM, 56 nM) and (70 nM, 37 nM) respectively and showed (EGFRwt/EGFRT790M S.I.) of (range: 4.44-9.40) compared to erlotinib (2.03) and osmertinib (1.89).

Enantiomeric separation of thiourea derivatives of naringenin on amylose and cellulose polymeric chromatographic chiral columns.

Due to a great demand for amylose and cellulose polymeric chromatographic chiral columns, the enantiomeric separation of thiourea derivatives of naringenin was achieved on the different amylose (Chiralpak-IB) and cellulose chiral (Chiralcel-OJ and Chiralcel-OD-3R) columns with varied chromatographic conditions. The isocratic mobile phases used were ethanol and methanol, where ethanol/hexane and methanol/hexane were used as gradient mode and were prepared in volume/volume relation. The separation and resolution factors for all the enantiomers were in the range of 1.25 to 3.47 and 0.48 to 1.75, respectively. The enantiomeric resolution was obtained within 12 min making fast separation. The docking studies confirmed the chiral recognition mechanisms with binding affinities in the range of -4.7 to -5.7 kcal/mol. The reported compounds have good anticoagulant activities and may be used as anticoagulants in the future. Besides, chiral separation is fast and is useful for enantiomeric separation in any laboratory in the world.

Lysosomal BK channels facilitate silica-induced inflammation in macrophages.

BACKGROUND: Lysosomal ion channels are proposed therapeutic targets for a number of diseases, including those driven by NLRP3 inflammasome-mediated inflammation. Here, the specific role of the lysosomal big conductance Ca2+-activated K+ (BK) channel was evaluated in a silica model of inflammation in murine macrophages. A specific-inhibitor of BK channel function, paxilline (PAX), and activators NS11021 and NS1619 were utilized to evaluate the role of lysosomal BK channel activity in silica-induced lysosomal membrane permeabilization (LMP) and NLRP3 inflammasome activation resulting in IL-1ß release. METHODS: Murine macrophages were exposed in vitro to crystalline silica following pretreatment with BK channel inhibitors or activators and LMP, cell death, and IL-1ß release were assessed. In addition, the effect of PAX treatment on silica-induced cytosolic K+ decrease was measured. Finally, the effects of BK channel modifiers on lysosomal pH, proteolytic activity, and cholesterol transport were also evaluated. RESULTS: PAX pretreatment significantly attenuated silica-induced cell death and IL-1ß release. PAX caused an increase in lysosomal pH and decrease in lysosomal proteolytic activity. PAX also caused a significant accumulation of lysosomal cholesterol. BK channel activators NS11021 and NS1619 increased silica-induced cell death and IL-1ß release. BK channel activation also caused a decrease in lysosomal pH and increase in lysosomal proteolytic function as well as a decrease in cholesterol accumulation. CONCLUSION: Taken together, these results demonstrate that inhibiting lysosomal BK channel activity with PAX effectively reduced silica-induced cell death and IL-1ß release. Blocking cytosolic K+ entry into the lysosome prevented LMP through the decrease of lysosomal acidification and proteolytic function and increase in lysosomal cholesterol.

Design, synthesis, and biological evaluation of (thio)urea derivatives as potent Escherichia coli ß-glucuronidase inhibitors.

EcGUS has drawn considerable attention for its role as a target in alleviating serious GIAEs. In this study, a series of 72 (thio)urea derivatives were designed, synthesised, and biologically assayed. The bioassay results revealed that E-9 (IC50 = 2.68 µM) exhibited a promising inhibitory effect on EcGUS, surpassing EcGUS inhibitor D-saccharic acid-1,4-lactone (DSL, IC50 = 45.8 µM). Additionally, the inhibitory kinetic study indicated that E-9 (Ki = 1.64 µM) acted as an uncompetitive inhibitor against EcGUS. The structure-activity relationship revealed that introducing an electron-withdrawing group into the benzene ring at the para-position is beneficial for enhancing inhibitory activity against EcGUS. Furthermore, molecular docking analysis indicated that E-9 has a strong affinity to EcGUS by forming interactions with residues Asp 163, Tyr 472, and Glu 504. Overall, these results suggested that E-9 could be a potent EcGUS inhibitor, providing valuable insights and guidelines for the development of future inhibitors targeting EcGUS.

Sulfonyl thioureas with a benzo[d]thiazole ring as dual acetylcholinesterase/butyrylcholinesterase and human monoamine oxidase A and B inhibitors: An in vitro and in silico study.

A series of sulfonyl thioureas 6a-q containing a benzo[d]thiazole ring with an ester functional group was synthesized from corresponding substituted 2-aminobenzo[d]thiazoles 3a-q and p-toluenesulfonyl isothiocyanate. They had remarkable inhibitory activity against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), monoamine oxidase (MAO)-A, and MAO-B. Among thioureas, several compounds had notable activity in the order of 6k > 6 h > 6c (AChE), 6j > 6g > 6k (BChE), 6k > 6g > 6f (MAO-A), and 6i > 6k > 6h (MAO-B). Compound 6k was an inhibitor of interest due to its potent or good activity against all studied enzymes, with IC50 values of 0.027 ± 0.008 µM (AChE), 0.043 ± 0.004 µM (BChE), 0.353 ± 0.01 µM (MAO-A), and 0.716 ± 0.02 µM (MAO-B). This inhibitory capacity was comparable to that of the reference drugs for each enzyme. Kinetic studies of two compounds with potential activity, 6k (against AChE) and 6j (against BChE), had shown that both 6k and 6j followed competitive-type enzyme inhibition, with Ki constants of 24.49 and 12.16 nM, respectively. Induced fit docking studies for enzymes 4EY7, 7BO4, 2BXR, and 2BYB showed active interactions between sulfonyl thioureas of benzo[d]thiazoles and the residues in the active pocket with ligands 6k, 6i, and 6j, respectively. The stability of the ligand-protein complexes while each ligand entered the active site of each enzyme (4EY7, 7BO4, 2BXR, or 2BYB) was confirmed by molecular dynamics simulations.

2-Thioxo-3,4-dihydropyrimidine and thiourea endowed with sulfonamide moieties as dual EGFRT790M and VEGFR-2 inhibitors: Design, synthesis, docking, and anticancer evaluations.

The effectiveness of a new series of thiopyrimidine and thiourea containing sulfonamides moieties was tested on HCT-116, MCF-7, HepG2, and A549. HepG2 cell line was the one that all the new derivatives affected the most. The greatest potent compounds against the four HepG2, HCT116, MCF-7, and A549 cell lines were 8f and 8g with IC50 = 4.13, 6.64, 5.74, 6.85 µM and 4.09, 4.36, 4.22, 7.25 µM correspondingly. Compound 8g exhibited higher activity than sorafenib against HCT116 and MCF-7 but exhibited lower activity against HepG2 and A549. Moreover, compounds 8f and 8g exhibited higher activities than erlotinib on HepG2, HCT116, and MCF-7 but demonstrated lower activity on A549. The most potent cytotoxic derivatives 6f, 6g, 8c, 8d, 8e, 8f, and 8g were examined on normal VERO cell lines. Our derivatives have low toxicity on VERO cells with IC50 values ranging from 32.05 to 53.15 µM. Additionally, all compounds were assessed for dual VEGFR-2 and EGFRT790M inhibition effects. Compounds 8f and 8g were the most potent derivatives inhibited VEGFR-2 at IC50 value of 0.88 and 0.90 µM, correspondingly. As well, derivatives 8f and 8g could inhibit EGFRT790M demonstrating strongest effects with IC50 = 0.32 and 0.33 µM sequentially. Additionally, the greatest active derivatives ADMET profile was evaluated in relationship with sorafenib and erlotinib as reference agents. The data attained from docking were greatly related to that achieved from the biological testing.

Inhibition of the Sodium-Calcium Exchanger Reverse Mode Activity Reduces Alcohol Consumption in Rats.

Excessive and uncontrolled consumption of alcohol can cause alcohol use disorder (AUD), but its pharmacological mechanisms are not fully understood. Inhibiting the reverse mode activity of the sodium-calcium exchanger (NCX) can reduce the risk of alcohol withdrawal seizures, suggesting that NCX could play a role in controlling alcohol consumption. Here, we investigated how two potent inhibitors of NCX reverse mode activity, SN-6 (NCX1) and KB-R7943 (NCX3), affect voluntary alcohol consumption in adult male and female rats using the intermittent alcohol access two-bottle choice paradigm. Initially, animals were trained to drink 7.5% ethanol and water for four weeks before administering SN-6 and KB-R7934. Afterward, their alcohol intake, preference, and water intake were recorded 2 and 24 h after exposure to water and 7.5% ethanol. SN-6 significantly reduced alcohol consumption by 48% in male and 36% in female rats without affecting their water intake. Additionally, SN-6 significantly reduced alcohol preference in females by 27%. However, KB-R7943 reduced alcohol consumption by 42% in female rats and did not affect alcohol preference or water intake. These findings suggest that alcohol exposure increased NCX reverse activity, and targeting NCX1 could be an effective strategy for reducing alcohol consumption in subjects susceptible to withdrawal seizures.

Synthesis, Computational Study, and In Vitro α-Glucosidase Inhibitory Action of Thiourea Derivatives Based on 3-Aminopyridin-2(1H)-Ones.

Reactions with allyl-, acetyl-, and phenylisothiocyanate have been studied on the basis of 3-amino-4,6-dimethylpyridine-2(1H)-one, 3-amino-4-phenylpyridine-2-one, and 3-amino-4-(thiophene-2-yl)pyridine-2(1H)-one (benzoyl-)isothiocyanates, and the corresponding thioureide derivatives 8-11a-c were obtained. Twelve thiourea derivatives were obtained and studied for their anti-diabetic activity against the enzyme α-glucosidase in comparison with the standard drug acarbose. The comparison drug acarbose inhibits the activity of α-glucosidase at a concentration of 15 mM by 46.1% (IC50 for acarbose is 11.96 mM). According to the results of the conducted studies, it was shown that alkyl and phenyl thiourea derivatives 8,9a-c, in contrast to their acetyl-(benzoyl) derivatives and 10,11a-c, show high antidiabetic activity. Thus, 1-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)-3-phenylthiourea 9a has the highest inhibitory activity against the enzyme α-glucosidase, exceeding the activity of the comparison drug acarbose, which inhibits the activity of α-glucosidase by 56.6% at a concentration of 15 mm (IC50 = 9,77 mM). 1-(6-methyl-2-oxo 4-(thiophen-2-yl)-1,2-dihydropyridin-3-yl)-3-phenylthiourea 9c has inhibitory activity against the enzyme α-glucosidase, comparable to the comparison drug acarbose, inhibiting the activity of α-glucosidase at a concentration of 15 mm per 41.2% (IC50 = 12,94 mM). Compounds 8a, 8b, and 9b showed inhibitory activity against the enzyme α-glucosidase, with a lower activity compared to acarbose, inhibiting the activity of α-glucosidase at a concentration of 15 mM by 23.3%, 26.9%, and 35.2%, respectively. The IC50 against α-glucosidase for compounds 8a, 8b, and 9b was found to be 16.64 mM, 19.79 mM, and 21.79 mM, respectively. The other compounds 8c, 10a, 10b, 10c, 11a, 11b, and 11c did not show inhibitory activity against α-glucosidase. Thus, the newly synthesized derivatives of thiourea based on 3-aminopyridine-2(1H)-ones are promising candidates for the further modification and study of their potential anti-diabetic activity. These positive bioanalytical results will stimulate further in-depth studies, including in vivo models.

Design and Synthesis of Thiourea-Conjugating Organic Arsenic D-Glucose with Anticancer Activities.

Organic arsenic compounds such as p-aminophenylarsine oxide (p-APAO) are easier for structural optimization to improve drug-like properties such as pharmacokinetic properties, therapeutic efficacy, and target selectivity. In order to strengthen the selectivity of 4-(1,3,2-dithiarsinan-2-yl) aniline 7 to tumor cell, a thiourea moiety was used to strengthen the anticancer activity. To avoid forming a mixture of α/ß anomers, the strategy of 2-acetyl's neighboring group participation was used to lock the configuration of 2,3,4,6-tetra-O-acetyl-ß-d-glucopyranosyl isothiocyanate from 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide. 1-(4-(1,3,2-dithiarsinan-2-yl) aniline)-2-N-(2,3,4,6-tetra-O-acetyl-ß-d-glucopyranos-1-yl)-thiourea 2 can increase the selectivity of human colon cancer cells HCT-116 (0.82 ± 0.06 µM vs. 1.82 ± 0.07 µM) to human embryonic kidney 293T cells (1.38 ± 0.01 µM vs. 1.22 ± 0.06 µM) from 0.67 to 1.68, suggesting a feasible approach to improve the therapeutic index of arsenic-containing compounds as chemotherapeutic agents.

Design, Synthesis, Computational Studies, and Anti-Proliferative Evaluation of Novel Ethacrynic Acid Derivatives Containing Nitrogen Heterocycle, Urea, and Thiourea Moieties as Anticancer Agents.

In the present work, the synthesis of new ethacrynic acid (EA) derivatives containing nitrogen heterocyclic, urea, or thiourea moieties via efficient and practical synthetic procedures was reported. The synthesised compounds were screened for their anti-proliferative activity against two different cancer cell lines, namely, HL60 (promyelocytic leukaemia) and HCT116 (human colon carcinoma). The results of the in vitro tests reveal that compounds 1-3, 10, 16(a-c), and 17 exhibit potent anti-proliferative activity against the HL60 cell line, with values of the percentage of cell viability ranging from 20 to 35% at 1 µM of the drug and IC50 values between 2.37 µM and 0.86 µM. Compounds 2 and 10 showed a very interesting anti-proliferative activity of 28 and 48% at 1 µM, respectively, against HCT116. Two PyTAP-based fluorescent EA analogues were also synthesised and tested, showing good anti-proliferative activity. A test on the drug-likeness properties in silico of all the synthetised compounds was performed in order to understand the mechanism of action of the most active compounds. A molecular docking study was conducted on two human proteins, namely, glutathione S-transferase P1-1 (pdb:2GSS) and caspase-3 (pdb:4AU8) as target enzymes. The docking results show that compounds 2 and 3 exhibit significant binding modes with these enzymes. This finding provides a potential strategy towards developing anticancer agents, and most of the synthesised and newly designed compounds show good drug-like properties.