Small Molecule SHP2 Inhibitor LXQ-217 Affects Lung Cancer Cell Proliferation in Vitro and in Vivo.

BACKGROUND: SHP2 is highly expressed in a variety of cancer and has emerged as a potential target for cancer therapeutic agents. The identification of uncharged pTyr mimics is an important direction for the development of SHP2 orthosteric inhibitors. METHODS: Surface plasmon resonance analysis and cellular thermal shift assay were employed to verify the direct binding of LXQ-217 to SHP2. The inhibitory effect of LXQ-217 was characterized by linear Weaver-Burke enzyme kinetic analysis and BIOVIA Discovery Studio. The inhibition of tumor cell proliferation by LXQ-217 was characterized by cell viability assay, colony formation assays and hoechst 33258 staining. The inhibition of lung cancer proliferation in vivo was studied in nude mice after oral administration of LXQ-217. RESULTS: An electroneutral bromophenol derivative, LXQ-217, was identified as a competitive SHP2 inhibitor. LXQ-217 induced apoptosis and inhibited growth of human pulmonary epithelial cells by affecting the RAS-ERK and PI3 K-AKT signaling pathways. Long-term oral administration of LXQ-217 significantly inhibited the proliferation ability of lung cancer cells in nude mice. Moreover, mice administered LXQ-217 orally at high doses exhibited no mortality or significant changes in vital signs. CONCLUSIONS: Our findings on the uncharged orthosteric inhibitor provide a foundation for further development of a safe and effective anti-lung cancer drug.

Synthesis and Biological Activity of Some Bromophenols and Their Derivatives Including Natural Products.

In addition to the first synthesis of the natural bromophenol butyl 2-(3,5-dibromo-4-hydroxyphenyl)acetate (1), indene derivatives 34 and 35 were synthesized from 3-phenylpropenal derivatives in BBr3 medium. Five known natural bromophenols and some derivatives were synthesized by known methods. Cholinesterase (ChEs) inhibitors reduce the breakdown of acetylcholine and are used in the treatment of Alzheimer's disease (AD) and dementia symptoms. The inhibition effects of all obtained compounds were examined towards acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and α-glycosidase enzymes. All synthesized compounds demonstrated the strong inhibition effects against both cholinergic enzymes. For determination of Ki values of novel bromophenols Lineweaver-Burk graphs were obtained. Ki values were found in the ranging of 0.13-14.74 nM for AChE, 5.11-23.95 nM for BChE, and 63.96-206.78 nM for α-glycosidase, respectively. All bromophenols and their derivatives exhibit effective inhibition profile when compared to positive controls.

A Novel Bromophenol Compound from Leathesia nana Inhibits Breast Cancer in a Direct Tumor Killing and Immunotherapy Manner.

Considering the resistance and toxicity of traditional chemotherapeutic drugs, seeking potential candidate for treating breast cancer effectively is a clinical problem that should be solved urgently. Natural products have attracted extensive attention, owing to their multi-target advantages and low toxicity. In the current study, the effects of XK-81, a novel bromophenol compound extracted from Leathesia nana, on breast cancer, and its underlying mechanisms, were explored. Firstly, data from in vitro experiments indicated that 4T-1, one of common mouse breast cancer cell lines, was a XK-81-susceptible cell line, and ferroptosis was the major death manner in response to XK-81 treatment, which was evidenced by increasing intracellular Fe2+ and ROS level with condensed mitochondrial membrane densities, as well as decreasing the protein expressions of SLC7A11 and GPX4. In vivo, XK-81 suppressed the growth of 4T-1 breast-tumor in both BALB/C mice and zebrafish. Obviously, XK-81 decreased the protein expression of SLC7A11 and GPX4 in tumor tissues, hinting at the occurrence of ferroptosis. Moreover, XK-81 increased CD8+ T cells and NK cells numbers and regulated M1/M2 macrophage ratio in tumor tissues, indicating XK-81's immunotherapeutic effect. Additionally, the secretions of immune-related cytokines, including TNF-α, IL-1ß, and IL-12, were elevated with XK-81 stimulation in RAW 264.7 cells. Intriguingly, compared with doxorubicin-induced heart damage, XK-81 demonstrated the therapeutic advantage of little cardiotoxicity on the heart. XK-81 demonstrated potential antitumor advantage by both directly inducing ferroptosis-mediated death of tumor cells and immunization.

Use of Euphorbia balsamifera Extract in Precursor Fabrication of Silver Nanoparticles for Efficient Removal of Bromocresol Green and Bromophenol Blue Toxic Dyes.

Silver nanoparticles (Ag-NPs) are attracting great attention for their use in various applications, along with methods for their green and facile production. In this study, we present a new eco-friendly approach based on the use of Euphorbia balsamifera extract (EBE) in the green synthesis of silver nanoparticles (Ag-NPs), which are then applied as a reducing and stabilizing agent for the efficient removal of water-based reactive dyes such as bromocresol green (BCG) and bromophenol blue (BPB). The as-prepared Ag-NPs are quasi-spherical in shape, with an average diameter of 20-34 nm. Diverse characterization methods, including X-ray diffractometry (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Brunauer-Emmett-Teller (BET) analysis, were used to analyze these Ag-NPs. The results reveal that water-soluble biomolecules in the Euphorbia balsamifera extract play an important role in the formation of the Ag-NPs. The removal of toxic dyes was studied under varied operational parameters such as Ag-NP dosage, initial dye concentration, pH, stirring time, and temperature. Under the optimum investigated conditions, nearly 99.12% and 97.25% of the bromocresol green and bromophenol blue dyes, respectively, were removed. Both BCG and BPB adsorption were found to adhere to pseudo-second-order kinetics (r22 = 1 and 0.995) and fit the Langmuir isotherm models well (R12 = 0.998 and 0.994), with maximal monolayer adsorption capacities of 20.40 and 41.03 mg/g, respectively. Their adsorption processes were observed to be intrinsically endothermic. The results confirm the potential of the Euphorbia balsamifera extract as a low-cost, nontoxic, and eco-friendly natural resource for the synthesis of Ag-NPs that may be useful in the remediation of hazardous dye-contaminated water sources.

Polyethylene glycol capped copper ferrite porous nanostructured materials for efficient photocatalytic degradation of bromophenol blue.

Three different types (blank, annealed, and functionalized) of copper ferrite nanoparticles (CuFe2O4) were synthesized by the co-precipitation method. The CuFe2O4 NPs were characterized by Fourier transform infrared (FTIR), Scanning electron microscopy (SEM), X-ray diffraction (XRD), and Energy-dispersive X-ray spectroscopy (EDX) techniques. FTIR analysis confirmed that 3-APTES is successfully grafted on the surface of CuFe2O4 NPs. XRD results show the amorphous nature of blank CuFe2O4 NPs, and crystalline structure was observed for annealed and functionalized CuFe2O4 NPs. XRD results revealed that crystallite size ranges from 23.6 to 34.6 nm. SEM micrographs of blank CuFe2O4 NPs show the irregular shape and size of the nanostructure. The spherical and strongly linked structure was seen in the micrograph of functionalized CuFe2O4 NPs. EDX analysis revealed the nanostructure composed of Fe, Cu, O, and a small percentage of Si. The photocatalytic degradation efficiency of synthesized CuFe2O4 NPs was examined under UV irradiation in an aqueous medium against bromophenol blue (BPB) dye. The effect of different parameters such as irradiation time and pH on the photodegradation of BPB dye was studied by all three types of CuFe2O4 photocatalyst. Results show that the maximum photocatalytic degradation efficiency was observed for functionalized CuFe2O4 nanoparticles that degraded 98% of BPB dye in the acidic medium at pH = 1. The optimum contact time for dye degradation was 120 min by synthesized photocatalyst. Photodegradation performance of blank and annealed CuFe2O4 NPs is less than 90%. The synthesized CuFe2O4 NPs were recycled and reused, which shows good photocatalytic degradation efficiency up to 4 consecutive cycles. The kinetic model displayed that degradation reaction followed pseudo 1st order kinetics. The blank, annealed, and functionalized CuFe2O4 NPs have turnover numbers of 10.7x10 (Mudhoo et al., 2019), 12.9x10 (Mudhoo et al., 2019), and 22.2x10 (Mudhoo et al., 2019) (kg-1 sec-1) accordingly. In conclusion, all results revealed the high efficiency of prepared photocatalyst for tested hazardous dye from wastewater and encouraged more work on photodegradation of organic pollutants from wastewater.

Amino Acid-Coupled Bromophenols and a Sulfated Dimethylsulfonium Lanosol from the Red Alga Vertebrata lanosa.

Vertebrata lanosa is a red alga that can commonly be found along the shores of Europe and North America. Its composition of bromophenols has been studied intensely. The aim of the current study was therefore to further investigate the phytochemistry of this alga, focusing more on the polar components. In total, 23 substances were isolated, including lanosol-4,7-disulfate (4) and the new compounds 3,5-dibromotyrosine (12), 3-bromo-5-sulfodihydroxyphenylalanine (13), 3-bromo-6-lanosyl dihydroxyphenylalanine (14), 3-(6'-lanosyl lanosyl) tyrosine (15) and 5-sulfovertebratol (16). In addition, 4-sulfo-7-dimethylsulfonium lanosol (7) was identified. While, in general, the dimethylsulfonium moiety is widespread in algae, its appearance in bromophenol is unique. Moreover, the major glycerogalactolipids, including the new ((5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid 3'-[(6''-O-α-galactopyranosyl-ß-D-galactopyranosyl)]-1-glycerol ester (23), and mycosporine-like amino acids, porphyra-334 (17), aplysiapalythine A (18) and palythine (19), were identified.

Co-metabolic biodegradation of 4-bromophenol in a mixture of pollutants system by Arthrobacter chlorophenolicus A6.

Brominated phenols are listed as priority pollutants together with nitrophenol and chlorophenol are the key components of paper pulp wastewater. However, the biodegradation of bromophenol in a mixed substrate system is very scanty. In the present investigation, simultaneous biodegradation kinetics of three substituted phenols 4-bromophenol (4-BP), 4-nitrophenol (4-NP), and 4-chlorophenol (4-CP) were investigated using Arthrobacter chlorophenolicus A6. A 23 full factorial design was applied with varying 4-BP and 4-CP from 75-125 mg/L and 4-NP from 50-100 mg/L. Almost complete degradation of this mixture of substituted phenols was achieved at initial concentration combinations of 125, 125, and 100 mg/L of 4-CP, 4-BP, and 4-NP, respectively, in 68 h. Statistical analysis of the results revealed that, among the three variables, 4-NP had the most prominent influence on the degradation of both 4-CP and 4-BP, while the concentration of 4-CP had a strong negative interaction effect on the biodegradation of 4-NP. Irrespective of the concentration levels of these three substrates, 4-NP was preferentially biodegraded over 4-CP and 4-BP. Furthermore, 4-BP biodegradation rates were found to be higher than those of 4-CP, followed by 4-NP. Besides, the variation of the biomass yield coefficient of the culture was investigated at different initial concentration combinations of these substituted phenols. Although the actinomycetes consumed 4-NP at a faster rate, the biomass yield was very poor. This revealed that the microbial cells were more stressed when grown on 4-NP compared to 4-BP and 4-CP. Overall, this study revealed the potential of A. chlorophenolicus A6 for the degradation of 4-BP in mixed substrate systems.

Synthesis and Aldose Reductase Inhibition Effects of Novel N-Benzyl-4-Methoxyaniline Derivatives.

In the current study, starting from 4-methoxyaniline, four Schiff bases were synthesized from benzaldehydes with Br and OMe. Corresponding N-benzylanilines and their derivatives were obtained from reductions (by NaBH4 ) and substitutions (by acyl and tosyl chlorides) of these bases, respectively. The inhibitory effects of the sixteen compounds, twelve of which were novel compounds are examined. Then, we conducted molecular docking and binary QSAR studies to determine inhibitory-enzyme interactions of compounds that show an inhibitory effect. Our results reveal that methoxyanilline-derived compounds show good biological activities. The most active compound (22) has IC50 values of 2.83 µM. These novel AR enzyme inhibitors may open new avenues for better AR inhibitors in the future.

Synthesis of Novel Bromophenol with Diaryl Methanes-Determination of Their Inhibition Effects on Carbonic Anhydrase and Acetylcholinesterase.

In this work, nine new bromophenol derivatives were designed and synthesized. The alkylation reactions of (2-bromo-4,5-dimethoxyphenyl)methanol (7) with substituted benzenes 8-12 produced new diaryl methanes 13-17. Targeted bromophenol derivatives 18-21 were synthesized via the O-Me demethylation of diaryl methanes with BBr3. Moreover, the synthesized bromophenol compounds were tested with some metabolic enzymes such as acetylcholinesterase (AChE), carbonic anhydrase I (CA I), and II (CA II) isoenzymes. The novel synthesized bromophenol compounds showed Ki values that ranged from 2.53 ± 0.25 to 25.67 ± 4.58 nM against hCA I, from 1.63 ± 0.11 to 15.05 ± 1.07 nM against hCA II, and from 6.54 ± 1.03 to 24.86 ± 5.30 nM against AChE. The studied compounds in this work exhibited effective hCA isoenzyme and AChE enzyme inhibition effects. The results show that they can be used for the treatment of glaucoma, epilepsy, Parkinson's as well as Alzheimer's disease (AD) after some imperative pharmacological studies that would reveal their drug potential.

Colorimetric detection of residual quaternary ammonium compounds on dry surfaces and prediction of antimicrobial activity using bromophenol blue.

Controlling and monitoring the residual activity of quaternary ammonium compounds (QACs) are critical for maintaining safe yet effective levels of these agents in the environment. This study investigates the utility of bromophenol blue (BPB) as a safe, rapid and user-friendly indicator to detect in situ residual QACs dried on hard, non-porous surfaces, as well a means to assess their antimicrobial efficacy. At pH 7, BPB has a purple colour which turns blue upon its complexation with QACs such as didecyldimethylammonium chloride (DDAC). BPB itself has no antimicrobial properties up to 400 ppm. Within the range of 0-400 ppm, BPB colour change was tied to specific DDAC antimicrobial performances with a detection threshold of 100 ppm. BPB concentration and application volume could be adjusted such that a colour shift from purple to blue correlated with a set percent reduction (>99·9%) in test bacteria (Staphylococcus aureus and Klebsiella aerogenes). The BPB solutions developed in this study yielded similar colour shifts on polycarbonate and stainless steel surfaces and did not cross-react with chemical ingredients commonly found in sanitizers and disinfectant products. Overall, this study suggests that BPB provides a simple solution to safely monitor the post-application level and biocidal activity of residual dried QACs on surfaces.

The Ovariol Morphology and Ultrastructure of Poecilimon ataturki Ünal, 1999 (Orthoptera, Tettigoniidae) and the Histochemical Features of the Yolk Granules.

This study presents the oocyte development of Poecilimon ataturki Ünal, 1999 (Orthoptera, Tettigoniidae) with histology, morphology, and histochemistry by using a stereomicroscope, a light microscope, a scanning electron microscope, and a transmission electron microscope. The ovary in this species is a panoistic type which contains many ovarioles which consist of terminal filament, germarium, and vitellarium. Germarium is the region that has undifferentiated cells which generate the oocytes and follicular cells. In the vitellarium region, yolk granules start to cover the whole oocyte. In histochemical studies, to determine the content of the yolk granules, proteins, and carbohydrates in oocytes were treated with a bromophenol blue (BPB) method, a mercury bromophenol blue (mBPB) method, and a periodic acid Schiff (PAS) method, respectively. As a result of these methods, the yolk granules gave positive results in ovariole sections treated with the PAS and the BPB, while the mBPB staining was negative.

A Dearomatization/Debromination Strategy for the [4+1] Spiroannulation of Bromophenols with α,ß-Unsaturated Imines.

A novel [4+1] spiroannulation of o- & p-bromophenols with α,ß-unsaturated imines has been developed for the direct synthesis of a new family of azaspirocyclic molecules. Notably, several other halophenols (X=Cl, I) were also applicable for this transformation. Moreover, a catalytic asymmetric version of the reaction was realized with 1-bromo-2-naphthols by using a chiral ScIII /Py-Box catalyst. Mechanistic studies revealed that this domino reaction proceeded through electrophile-triggered dearomatization of phenol derivatives at their halogenated positions and followed by halogen-displacement with N-nucleophiles via a radical-based SRN 1 mechanism.

The first synthesis, carbonic anhydrase inhibition and anticholinergic activities of some bromophenol derivatives with S including natural products.

Starting from vanillin, known four benzyl bromides with Br were synthesized. The first synthesis of natural product 3,4-dibromo-5-((methylsulfonyl)methyl)benzene-1,2-diol (2) and 3,4,6-tribromo-5-((methylsulfonyl)methyl)benzene-1,2-diol (3) and derivatives were carried out by demethylation, acetylatilation, oxidation and hydrolysis reactions of the benzyl bromides. Also, these compounds were tested against some important enzymes like acetylcholinesterase and butyrylcholinesterase enzymes, carbonic anhydrase I, and II isoenzymes. The novel bromophenols showed Ki values of in range of 53.75 ±â€¯12.54-234.68 ±â€¯46.76 nM against hCA I, 42.84 ±â€¯9.36 and 200.54 ±â€¯57.25 nM against hCA II, 0.84 ±â€¯0.12-14.63 ±â€¯3.06 nM against AChE and 0.93 ±â€¯0.20-18.53 ±â€¯5.06 nM against BChE. Induced fit docking process performed on the compounds inhibiting hCA I, hCA II, AChE, and BChE receptors. Hydroxyl group should exist at the aromatic ring of the compounds for inhibition of the enzymes. The moieties reported in this study will be useful for design of more potent and selective inhibitors against the enzymes.

Synthesis and biological evaluation of bromophenol derivatives with cyclopropyl moiety: Ring opening of cyclopropane with monoester.

Trans-(1R*,2R*,3R*)-Ethyl 2-(3,4-dimethoxyphenyl)-3-methylcyclopropane-1-carboxylate (6) and its cis isomer 7 were obtained from the reaction of the methyl isoeugenol (5) with ethyl diazoacetate. The reduction and bromination reactions of the ester 6 and 7 together with the hydrolysis of all esters were carried out. Opening ring of cyclopropane was observed in the reaction of 7 with bromine. The opening of cyclopropane ring with COOR and synthesis of esters, alcohols and acids (6-26) are new. These obtained bromophenol derivatives (6-26) were effective inhibitors of the cytosolic carbonic anhydrase I and II isoforms (hCA I and II) and acetylcholinesterase (AChE) enzymes with Ki values in the range of 7.8 ±â€¯0.9-58.3 ±â€¯10.3 nM for hCA I, 43.1 ±â€¯16.7-150.2 ±â€¯24.1 nM for hCA II, and 159.6 ±â€¯21.9-924.2 ±â€¯104.8 nM for AChE, respectively. Acetylcholinesterase inhibitors are the most popular drugs applied in the treatment of diseases such as Alzheimer's disease, Parkinson's disease, senile dementia, and ataxia, among others.

Degradation of 4-bromophenol by Ochrobactrum sp. HI1 isolated from desert soil: pathway and isotope effects.

Anthropogenic activities have introduced elevated levels of brominated phenols to the environment. These compounds are associated with toxic and endocrine effects, and their environmental fate is of interest. An aerobic strain Ochrobactrum sp. HI1 was isolated from soils in the vicinity of a bromophenol production plant and tested for its ability to degrade 4-bromophenol (4-BP). A ring hydroxylation pathway of degradation was proposed, using the evidence from degradation intermediates analysis and multi-element (C, Br, H) compound-specific isotope analysis. Benzenetriol and 4-bromocatechol were detected during degradation of 4-bromophenol. Degradation resulted in a normal carbon isotope effect (εC = -1.11 ± 0.09‰), and in insignificant bromine and hydrogen isotope fractionation. The dual C-Br isotope trend for ring hydroxylation obtained in the present study differs from the trends expected for reductive debromination or photolysis. Thus, the isotope data reported herein can be applied in future field studies to delineate aerobic biodegradation processes and differentiate them from other natural attenuation processes.

Enhanced detoxification of p-bromophenol by novel Zr/Ag-TiO2@rGO ternary composite: Degradation kinetics and phytotoxicity evolution studies.

The toxicity and persistence of the halogenated aromatics, particularly brominated phenolic compounds have drawn serious concerns to the environment, emphasizing the potential effects on human health and ecosystems balance. Advanced oxidation process (AOP) has received much attention as an alternative for the conventional wastewater treatment methods to treat water contaminated with toxic pollutants. This study investigated the degradation and detoxification of p-bromophenol (p-BP) by a novel Zr/Ag-TiO2@rGO photocatalyst under visible light. Upon 3 h of visible light irradiation over Zr/Ag-TiO2@rGO, more than 95% of p-BP (15 mg/L) degradation was achieved at a rate of 0.23 min-1. The degradation products were identified by GC-MS and possible degradation pathway was proposed. The phytotoxicity evolution of the degraded products was assessed on Vigna radiata (V. radiata), in which seeds treated with pure p-BP showed less germination (40%) compared to degradation products (100%). Furthermore, the germination index (GI) of p-BP was found to be 11.1% before degradation while it increased to 80.5% after 3 h of degradation indicated that this photodegradation process achieved detoxification of p-BP. Thus, this study demonstrated that p-BP elimination and detoxification could be simply achieved with Zr/Ag-TiO2@rGO nanocomposite under visible light irradiation, which provides new solution for wastewater treatment and water reuse in crop irrigation.

Discovery of meta-Amido Bromophenols as New Antitubercular Agents.

A series of meta-amido bromophenol derivatives were designed and synthesized. The compounds were found to potently inhibit the growth of Mycobacterium tuberculosis H37Ra. They also exhibited moderate inhibitory activity against Mycobacterium tuberculosis H37Rv and multidrug-resistant strains. The compounds did not show inhibitory activity against normal Gram-positive and Gram-negative bacteria. Moderate cytotoxicities and good metabolic stability were observed for the selected compounds. The results demonstrated meta-amido bromophenols as a new class of antitubercular agents with good potentials.

A Novel Bromophenol Derivative BOS-102 Induces Cell Cycle Arrest and Apoptosis in Human A549 Lung Cancer Cells via ROS-Mediated PI3K/Akt and the MAPK Signaling Pathway.

Bromophenol is a type of natural marine product. It has excellent biological activities, especially anticancer activities. In our study of searching for potent anticancer drugs, a novel bromophenol derivative containing indolin-2-one moiety, 3-(4-(3-([1,4'-bipiperidin]-1'-yl)propoxy)-3-bromo-5-methoxybenzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide (BOS-102) was synthesized, which showed excellent anticancer activities on human lung cancer cell lines. A study of the mechanisms indicated that BOS-102 could significantly block cell proliferation in human A549 lung cancer cells and effectively induce G0/G1 cell cycle arrest via targeting cyclin D1 and cyclin-dependent kinase 4 (CDK4). BOS-102 could also induce apoptosis, including activating caspase-3 and poly (ADP-ribose) polymerase (PARP), increasing the Bax/Bcl-2 ratio, enhancing reactive oxygen species (ROS) generation, decreasing mitochondrial membrane potential (MMP, ΔΨm), and leading cytochrome c release from mitochondria. Further research revealed that BOS-102 deactivated the PI3K/Akt pathway and activated the mitogen-activated protein kinase (MAPK) signaling pathway resulting in apoptosis and cell cycle arrest, which indicated that BOS-102 has the potential to develop into an anticancer drug.

Antidiabetic potential: In vitro inhibition effects of bromophenol and diarylmethanones derivatives on metabolic enzymes.

Aldose reductase converts glucose to sorbitol in the polyol pathway. It is an important enzyme to prevent diabetic complications. In this study, we studied the inhibitory effects of bromophenol derivatives on aldose reductase (AR), α-glucosidase, and α-amylase enzymes. In the bromophenols series, compound 1f showed the maximum inhibition effect against AR with a Ki value of 0.05 ± 0.01 µM, while compound 1d showed the lowest inhibition effect against AR with a Ki value of 1.13 ± 0.99 µM. In addition, α-amylase from porcine pancreas and α-glucosidase from Saccharomyces cerevisiae were used as enzymes. In this study, all compounds were tested for the inhibition of the α-glucosidase enzyme and demonstrated efficient inhibition profiles with Ki values in the range of 43.62 ± 5.28 to 144.37 ± 16.37 nM against α-glucosidase. Additionally, these compounds were tested against the α-amylase enzyme, which determined an effective inhibition profile with IC50 values in the range of 9.63-91.47 nM. These compounds can be selective inhibitors of AR, α-glucosidase, and α-amylase enzymes as antidiabetic agents.

Carbonic anhydrase inhibitor acetazolamide shifts synaptic vesicle recycling to a fast mode at the mouse neuromuscular junction.

Acetazolamide (AZ), a molecule frequently used to treat different neurological syndromes, is an inhibitor of the carbonic anhydrase (CA), an enzyme that regulates pH inside and outside cells. We combined fluorescent FM styryl dyes and electrophysiological techniques at ex vivo levator auris longus neuromuscular junctions (NMJs) from mice to investigate the modulation of synaptic transmission and vesicle recycling by AZ. Transmitter release was minimally affected by AZ, as evidenced by evoked and spontaneous end-plate potential measurements. However, optical evaluation with FM-styryl dyes of vesicle exocytosis elicited by 50 Hz stimuli showed a strong reduction in fluorescence loss in AZ treated NMJ, an effect that was abolished by bathing the NMJ in Hepes. The remaining dye was quenched by bromophenol, a small molecule capable of diffusing inside vesicles. Furthermore, in transgenic mice expressing Synaptophysin-pHluorin (SypHy), the fluorescence responses of motor nerve terminals to a 50 Hz train of stimuli was decrease to a 50% of controls in the presence of AZ. Immunohistochemistry experiments to evaluate the state of the Myosin light chain kinase (MLCK), an enzyme involved in vesicle recycling, demonstrated that MLCK phosphorylation was much stronger in the presence than AZ than in its absence in 50 Hz stimulated NMJs. We postulate that AZ, via cytosol acidification and activation of MLCK, shifts synaptic vesicle recycling to a fast (kiss-and-run) mode, which changes synaptic performance. These changes may contribute to the therapeutic action reported in many neurological syndromes like ataxia, epilepsy, and migraine.