Microwave-Assisted Organic Synthesis,structure–activity relationship,kinetics and molecular docking studies of non-cytotoxic benzamide derivatives as selective butyrylcholinesterase inhibitors

A series of benzamide derivatives 1–12 with various functional groups (–H, –Br, –F, –OCH₃, –OC₂H₅, and –NO₂) was synthesized using an economic, and facile Microwave-Assisted Organic Synthesis, and evaluated for acetylcholinesterase (ACHE) and butyrylcholinesterase (BCHE) activity in vitro. Structure–activity relationship showed that the substitution of –Br group influenced the inhibitory activity against BCHE enzyme. Synthesized compounds were found to be selective inhibitors of BCHE. In addition, all compounds 1–12 were found to be non-cytotoxic, as compared to the standard cycloheximide (IC₅₀ = 0.8 ± 0.2 µM). Among them, compound 3 revealed the most potent BCHE inhibitory activity (IC₅₀ = 0.8 ± 0.6 µM) when compared with the standard galantamine hydrobromide (IC₅₀ = 40.83 ± 0.37 µM). Enzyme kinetic studies indicated that compounds 1, 3–4, and 7–8 showed a mixed mode of inhibition against BCHE, while compounds 2, 5–6 and 9 exhibited an uncompetitive pattern of inhibition. Molecular docking studies further highlighted the interaction of these inhibitors with catalytically important amino acid residues, such as Glu197, Hip438, Phe329, and many others.     

Induction of heme oxygenase-1 by chamomile protects murine macrophages against oxidative stress

AimsProtection of cells from oxidative insult may be possible through direct scavenging of reactive oxygen species, or through stimulation of intracellular antioxidant defense mechanisms by induction of antioxidant gene expression. In this study we investigated the cytoprotective effect of chamomile and elucidated the underlying mechanisms.Main methodsThe cytoprotective effect of chamomile was examined on H₂O₂-induced cellular stress in RAW 264.7 murine macrophages.Key findingsRAW 264.7 murine macrophages treated with chamomile were protected from cell death caused by H₂O₂. Treatment with 50 μM H₂O₂ for 6 h caused significant increase in cellular stress accompanied by cell death in RAW 264.7 macrophages. Pretreatment with chamomile at 10–20 μg/mL for 16 h followed by H₂O₂ treatment protected the macrophages against cell death. Chamomile exposure significantly increased the expression of antioxidant enzymes viz. heme oxygenase-1 (HO-1), peroxiredoxin-1 (Prx-1), and thioredoxin-1 (Trx-1) in a dose-dependent manner, compared with their respective controls. Chamomile increased nuclear translocation of Nrf2 with increased phosphorylated Nrf2 levels, and binding to the antioxidant response element in the nucleus.SignificanceThese molecular findings for the first time provide insights into the mechanisms underlying the induction of phase 2 enzymes through the Keap1-Nrf2 signaling pathway by chamomile, and provide evidence that chamomile possesses antioxidant and cytoprotective properties.     

SERINE proteinase like activity in apolipophorin III from the hemolymph of desert locust, Schistocerca gregaria

Apolipophorin III (apoLp-III) has been known as a lipid transport protein of insects. Recent studies indicated the involvement of apoLp-III in immune reactions and in the control of cell destruction, but no enzymatic activity has so far been detected. In the present study, a protease from the hemolymph of Schistocerca gregaria was purified to homogeneity and its enzymatic activity was examined. Identity as chymotrypsin-like proteinase was established by its high affinity toward bulky aromatic substrates and its catalytic specificity for amide or ester bonds on the synthetic substrates, Suc-Ala-Ala-Pro-Xaa-AMC (where Xaa was Phe, Tyr, Trp, and Lys, and AMC is 7-amino-4-methyl-coumarin) and thiolbenzyl ester substrate Suc-Ala-Ala-Pro-Phe-SBzl. The sensitivity for serine protease and chymotrypsin-specific covalent inhibitors, PMSF, TPCK, and noncovalent inhibitors SGCI, showed that it is a chymotrypsin-like proteinase. It showed its maximum activity at pH 8.0 and 55°C for the hydrolysis of Suc-Ala-Ala-Pro-Tyr-AMC. According to similarities in the amino terminal sequence, molar mass (19 kDa) and retention on reversed-phase analytical high-performance liquid chromatography (HPLC) column, this protein is S. gregaria homologue of Locusta migratoria apoLp-III. Our data suggest that apoLp-III also has an inherent proteolytic activity. Results indicated that S. gregaria apoLp-III is a good catalyst and could be used as a biotechnological tool in food processing and in agricultural biotechnology.     

Determination of Arsenic in Scalp Hair of Children and its Correlation with Drinking Water in Exposed Areas of Sindh Pakistan

This study was focused on the analysis of arsenic (As) levels in scalp hair of children (age, <10 years) collected from two towns of Khairpur, Pakistan, to evaluate the effects of As-contaminated groundwater. For comparative purposes, scalp hair samples of children were also collected from that area having low levels of As (<10 μg/L) in drinking water. Groundwater and scalp hair samples of children were collected and analyzed by electrothermal atomic absorption spectrometry prior to microwave-assisted acid digestion. The average As concentrations in groundwater samples of two towns, Thari Mirwah and Gambat, were found to be 28.5 and 98.3 μg/L, respectively. The range of As concentrations in scalp hair samples of children who belong to Thari Mirwah and Gambat was 1.25-1.61 μg/g and 1.73-3.63 μg/g, respectively. Twenty percent of the total children who belong to Gambat have skin lesions on their hands and feet. A positive correlation coefficient (R = 0.91-0.99) was obtained between As contents in drinking water and scalp hairs of children of both towns.     

Determination of Arsenic Scalp Hair of Pakistani Children and Drinking Water for Environmental Risk Assessment

This cross-sectional study measured the arsenic level in scalp hair samples of children from two age groups exposed to contaminated underground water in Pakistan. The As concentrations in water and scalp hair samples were analyzed by electrothermal atomic absorption spectrometry. The studied areas were divided into three regions based on concentration of As in water and scalp hair of children who had been exposed to different levels of As through drinking water. The average As concentrations in underground water samples of sub-districts Faiz Ganj, Thari Mirwah, and Gambat were observed to be 15.2, 28.5, and 98.3 μg/L, respectively, which were marked as less, medium, and highly contaminated areas, respectively. The proportion of the children with lower body mass index significantly increased with increasing As exposure level; the dose–response relationship was consistently observed among the subgroups. The range of As concentrations in scalp hair samples of children of age 1–5 and 6–10 years was 0.21–3.88 and 0.32–3.63 μg/g, respectively. Thus, it is likely that children in our study areas are chronically exposed to As through drinking groundwater, suggesting that there is an urgent need to take immediate solution, especially in the Gambat sub-district.     

Hepatocellular carcinoma: targeting of oncogenic signaling networks in TRAIL resistant cancer cells

Apoptotic response in hepatocellular carcinoma (HCC) cells is impaired because of interconnectivity of proteins into complexes and signaling networks that are highly divergent in time and space. TNF-related apoptosis-inducing ligand (TRAIL) has emerged as an attractive anticancer agent reported to selectively induce apoptosis in cancer cells. Although diametrically opposed roles of TRAIL are reported both as an inducer of apoptosis and regulator of metastasis, overwhelmingly accumulating experimental evidence highlighting apoptosis inducing activity of TRAIL is directing TRAIL into clinical trials. Insights from TRAIL mediated signaling in HCC research are catalyzing new lines of study that should not only explain molecular mechanisms of disease but also highlight emerging paradigms in restoration of TRAIL mediated apoptosis in resistant cancer cells. It is becoming progressively more understandable that phytochemicals derived from edible plants have shown potential in modelling their interactions with their target proteins. Rapidly accumulating in vitro and in-vivo evidence indicates that phytonutrients have anticancer activity in rodent models of hepatocellular carcinoma. In this review we bring to limelight how phytonutrients restore apoptosis in hepatocellular carcinoma cells by rebalancing pro-apoptotic and anti-apoptotic proteins. Evidence has started to emerge, that reveals how phytonutrients target pharmacologically intractable proteins to suppress cancer. Target-based small-molecule discovery has entered into the mainstream research in the pharmaceutical industry and a better comprehension of the genetics of patients will be essential for identification of responders and non-responders.     

Molecular Diagnosis of Fragile X Syndrome in Subjects with Intellectual Disability of Unknown Origin: Implications of Its Prevalence in Regional Pakistan

Fragile-X syndrome (FXS) is the most common form of inherited intellectual disability (ID) and affects 0.7–3.0% of intellectually compromised population of unknown etiology worldwide. It is mostly caused by repeat expansion mutations in the FMR1 at chromosome Xq27.3. The present study aimed to develop molecular diagnostic tools for a better detection of FXS, to assess implementation of diagnostic protocols in a developing country and to estimate the prevalence of FXS in a cohort of intellectually disabled subjects from Pakistan. From a large pool of individuals with below normal IQ range, 395 subjects with intellectual disability of unknown etiology belonging to different regions of the country were recruited. Conventional-PCR, modified-PCR and Southern blot analysis methods were employed for the detection of CGG repeat polymorphisms in the FMR1 gene. Initial screening with conventional-PCR identified 13 suspected patients. Subsequent investigations through modified PCR and Southern blot analyses confirmed the presence of the FMR1 mutation, suggesting a prevalence of 3.5% and 2.8% (mean 3.3%) among the male and female ID patients, respectively. These diagnostic methods were further customized with the in-house conditions to offer robust screening of referral patients/families for diagnostics and genetic counseling. Prescreening and early diagnosis are crucial for designing a prudent strategy for the management of subjects with ID. Outcome of the study recommends health practitioners for implementation of molecular based FXS diagnosis in routine clinical practice to give a better care for patients similar to the ones included in the study.