Synthesis and activity of hydroxyethylene peptidomimetic inhibitors of humanβ-secretase

A series of β-secretase peptidomimetic inhibitors with Leu＊Ala hydroxyethylene dipeptide isostere were synthesized and their β-secretase inhibitory activities were measured. The most potent compound N9 showed an inhibitory rate of 59.66% （10 mg/mL）. Compound N9 might be further modified by means of computational chemical methodology.

Synthesis of Hydroxyethylene-based β-Secretase Inhibitors

Aim To discuss in depth the synthesis of hydroxyethylene dipeptide-based β-secretase inhibitors; Methods Organic reactions such as nucleophilic addition and substitution assisted by organometallic agents, catalytic hydrogenation, and classic peptide coupling were used to synthesize peptidomimetic β-secretase inhibitors. Results Ideal reaction conditions and potential problems were investigated, and one of the designed β-secretase inhibitors 13 （as a model） was synthesized successfully; Conclusion This approach might be used to build up the β-secretase inhibitor library and to search for new molecular candidates.

Divalent cation tolerance protein binds to β-secretase and inhibits the processing of amyloid precursor protein

The deposition of amyloid-beta is a pathological hallmark of Alzheimer＇s disease, Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase （beta-site amyloid precursor protein-cleaving enzyme 1） and r-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of AIzheimer＇s disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1- interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer＇s disease.

Design, Synthesize and Bio-Evaluate 1,2-Dihydroisoquinolin-3(4H)-One Derivates as Acetylcholinesterase and β-Secretase Dual Inhibitors in Treatment with Alzheimer’s Disease

With the recent research advances in molecular biology and technology, many credible hypothe-ses about the progress of Alzheimer’s disease (AD) have been proposed, among which the amyloid and cholinergic hypotheses are commonly used to develop reliable therapeutic agents. The multitarget-directed ligand (MTDL) approach was taken in this work to develop multi-functional agents, which can mainly serve as dual BACE 1 and AChE inhibitors. Depending on the scaffolds of (+)-(S)- dihydro-ar-tumerone and (-)-gallocatechin gallate, 3 series of new compounds have been designed, synthesized and evaluated, from which we have identified 2-(2-(3-methylbenzoyl)-3-oxo-1,2,3,4- tetrahydroisoquinolin-6-yl) isoindoline-1,3-dione (3d) as a new cholinesterase and β-secretase dual inhibitor without toxicity. Furthermore, 3d also exhibits hydrogen peroxide scavenging activity which could help to reduce the reactive oxygen species (ROS) in the brain of AD patients.

Structure-based design of hexahydropyrimidin-5-ols as novel non-peptidic β-secretase inhibitors

Based upon the crystal structure of a previously reported fragment hit that binds to Corresponding author. β-secretase, a novel series of non-peptidic small-molecule β-secretase inhibitors, namely hexahydropyrimidin-5-ols, along with two series of their analogues, were rationally designed through structural modification. The CADD study was performed and revealed good expectation. Inhibitory activities of the corresponding structural cores were tested, which provided further support for our design approach.

Precise location of proton of beta-secretase for catalytic aspartates(Asp 32 and Asp 228)in Alzheimer’s patients

BACKGROUND：β-secretase （β-site APP cleavage rate-limiting enzyme, BACE） has been proposed as a promising therapeutic target for Alzheimer＇s disease （AD）. BACE inhibition reduces production of β-amyloid peptide （Aβ） and promotes neural regeneration. Two catalytic aspartates （Asp 32 and Asp 228） exist in a monoprotonated state in the active BACE site, but the precise proton location remains unclear.OBJECTIVE：To explore the entire process of BACE enzymatic hydrolysis using quantum chemistry calculations, and to identify the precise proton location for Asp 32 and Asp 228 during the enzymatic process.DESIGN, TIME AND SETTING：According to protonation state of BACE, four tautomers were designed and quantum chemistry calculations were performed at the Department of Human Anatomy, Zhongshan School of Medicine, Sun Yat-sen University, China between October 2008 and March 2009.MATERIALS：Hardware：linux workstation （Department of Equipment, Sun Yat-sen University, China）; software：QSITE, Glide, Maestro （Schrodinger LLC, USA）, MOPAC 2007 （CAChe Research LLC, USA）, Triton 4.0 （National Centre for Biomolecular Research, Czech Republic） were used.METHODS：Using crystal structures of BACE to build a catalytic model （enzyme, catalytic water, and substrate peptide EVNLAAEF） on the computer and superimposition, four BACE tautomers （32i, 320, 228i, and 2280） in the monoprotonated state were developed with Schrodinger package. Hybrid quantum mechanical/molecular mechanic （QM/MM） calculations were performed at the B3LYP density functional theory level to identify the precise proton location for the dyad aspartic residues （Asp 32 and Asp 228）. Using the most possible tautomer as the reactant, the entire enzymatic hydrolysis of substrate EVNL/AAEF was simulated at the semiempirical level.MAIN OUTCOME MEASURES：The precise proton location of was measured by analyzing co-planarities of 4 BACE tautorners （32i, 32o, 228i, and 2280） in the monoprotonated state, because the dihedral formed by the carboxyl oxygen atoms of the dyad aspartic residues. The transition state and the production state, as well as activation energies and reaction enthalpies, were measured by calculating geometric and energy changes during catalytic reaction of the system.RESULTS：In the 2280 BACE tautomer, the dihedral angle of the four oxygen atoms in the catalytic aspartates was 8.7°, which was the lowest of four tautomers. The lowest activation energy and highest reaction enthalpy （Ea = 216.30 kJ/mol, AH = 30.98 kJ/mol） were also found in 2280, among the four tautomers during the catalytic reaction. In addition, when the reaction proceeded to the transition state, followed by product generation, the proton location was reversed to the inner oxygen of Asp 32 （32i） from the outer oxygen of Asp 228 （228o）.CONCLUSION：Results demonstrated the mechanism of Aβ generation. At beginning of BACE catalytic reaction, the precise proton location was preferred on the outer oxygen of Asp 228 （228o）. In this protonation state, catalytic reaction can proceed smoothly, with reduced active energy and heat release. When the reaction proceeded to the transition state and product generation, the proton location was reversed to the inner oxygen of Asp 32 （32i）. These results provide theoretical guidance for designing new drugs to protect neural cells and promote neural regeneration in Alzheimer＇s patients.

Effects of chronic cerebral hypoperfusion of beta- and gamma-secretase on learning and memory in rats

BACKGROUND： Chronic cerebral hypoxia and ischemia have been shown to be related to occurrence of sporadic Alzheimer＇s disease, and β- and y-secretase play an important role in the generation of β-amyloid protein. Early clinical symptoms in Alzheimer＇s disease patients include learning and memory deficits. OBJECTIVE： To measure learning and memory, as well as β- and β-secretase activities in the hippocampus of a cerebral ischemia/hypoxia rat model with chronic cerebral hypoperfusion. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Department of Pathology, Capital Medical University from March to December, 2008. MATERIALS： β- and y-secretase activity kits were purchased from R ＆ D Systems, USA. METHODS： Male Sprague Dawiey rats, aged 23 weeks, were randomly assigned to model （n = 56） and sham-surgery （n = 46） groups. Cerebral hypoperfusion rat models were established by bilateral common carotid occlusion. MAIN OUTCOME MEASURES： Morris water maze was used to test changes in escape latency and path length, and β- and y-secretase activities were measured on days 10, 30, 90, and 180 following surgery. RESULTS： Progressive cognitive impairment resulted from 30 days of chronic cerebral hypoperfusion, which lasted for 180 days after cerebral hypoperfusion. β-secretase activity was increased at 10 days after hypoperfusion, which continued until 180 days, with a 14.25% increase compared to the sham-surgery group; y-secretase activity was increased by 10.5%. CONCLUSION： Chronic cerebral hypoperfusion results in impaired spatial memory and upregulated β- and y-secretase activities, which could play an important role in β-amyloid production.

β-Secretase inhibitor increases amyloid-β precursor protein level in rat brain cortical primary neurons induced by okadaic acid

Background Senile plaques and neurofibrillary tangles （NFTs） represent two of the major histopathological hallmarks of Alzheimer＇s disease （AD）. The plaques are primarily composed of aggregated amyloid β （Aβ） peptides. The processing of amyloid-β precursor protein （AβPP） in okadaic acid （OA）-induced tau phosphorylation primary neurons was studied. Methods Primary cultures of rat brain cortical neurons were treated with OA and β-secretase inhibitor. Neurons＇ viability was measured. AβPP processing was examined by immunocytochemistry and Western blotting with specific antibodies against the AβPP-N-terminus （NT） and AβPP-C-terminus （CT）. Results Ten nmol/L OA had a time-dependent suppression effect on primary neurons＇ viability. The suppression effect was alleviated markedly by pretreatment with β-secretase inhibitor. After OA treatment, both AβPP and β-C-terminal fragment （βCTF） were significantly increased in neurons. AβPP level was increased further in neurons pretreated with β-secretase inhibitor. Conclusions In OA-induced tau phosphorylation cell model, inhibition of β-secretase may protect neurons from death induced by OA. Because of increased accumulation of AβPP in neurons after OA treatment, more AβPP turns to be cleaved by β-secretase, producing neurotoxic βCTF. As a potential effective therapeutic target, β-secretase is worth investigating further.

Purification of Full-Length <i>β</i>-Secretase Involved in Alzheimer’s Disease, and Proteomic Identification of Binding Partners

β-Secretase (BACE1 or β-site APP cleaving enzyme) is an acid protease that releases the neurotoxic 40 - 42 residue peptides (β-amyloid or A-β) from its glycoprotein precursor, (APP or amyloid precursor protein) which when released in brain is thought to give rise to cognitive decline in patients with Alzheimer’s Disease. Most structural studies on β-secretase have previously been performed with recombinant forms of the protease, in which the transmembrane coding region has been deleted. However, interactions with proteins of the same species are best studied using the full-length β-secretase as interactions are likely to be influenced by the hydrophobic nature and localization of its transmembrane regions. Here we develop a multi-step purification procedure that isolates a complex containing BACE1 from recombinant human cells using mild detergents in a procedure that retains other proteins within the complex and remains active in its β-site APP cleaving activity. Some of these proteins, eg reticulon 4, are identified by proteomics, and are known by previous studies performed by others to regulate the activity of BACE1 against APP. These interactions may aid the development of small proteins and peptides that could inhibit the release of aggregated forms of β-amyloid, and thus be useful therapeutically.

A GPCR/secretase complex regulates β- and γ-secretase specificity for Aβ production and contributes to AD pathogenesis

Dysregulation of β-site APP-cleaving enzyme （BACE） and/or γ-secretase leads to anomalous production of amyloid-β peptide （Aβ） and contributes to the etiology of Alzheimer＇s disease （AD）. Since these secretases mediate proteolytic processing of numerous proteins, little success has been achieved to treat AD by secretase inhibitors because of inevitable undesired side effects. Thus, it is of importance to unravel the regulatory mechanisms of these secretases. Here, we show that δ-opioid receptor （DOR） promotes the processing of Aβ precursor protein （APP） by BACE1 and γ-secretase, but not that of Notch, N-cadherin or APLP. Further investigation reveals that DOR forms a complex with BACE1 and γ-seeretase, and activation of DOR mediates the co-endocytic sorting of the secretases/ receptor complex for APP endoproteolysis. Dysfunction of the receptor retards the endocytosis of BACE1 and γ-secretase and thus the production of Aβ Consistently, knockdown or antagonization of DOR reduces secretase activities and ameliorates Aβ pathology and Aβ-dependent behavioral deficits, but does not affect the processing of Notch, N-cadherin or APLP in AD model mice. Our study not only uncovers a molecular mechanism for the formation of a DOR/secretase complex that regulates the specificity of secretase for Aβ production but also suggests that intervention of either formation or trafficking of the GPCR/secretase complex could lead to a new strategy against AD, potentially with fewer side effects.

Influence of Ginkgo Biloba extract on beta-secretase in rat hippocampal neuronal cultures following chronic hypoxic and hypoglycemic conditions

BACKGROUND： Preparation of Ginkgo leaf has been widely used to improve cognitive deficits and dementia, in particular in Alzheirner＇s disease patients. However, the precise mechanism of action of Ginkgo leaf remains unclear. OBJECTIVE： To explore the effect of Ginkgo Biloba extract （Egb761）, Ginaton, on β -secretase expression in rat hippocampal neuronal cultures following chronic hypoxic and hypoglycemic conditions. DESIGN, TIME AND SETTNG： Completely by randomized, grouping study. The experiment was performed at the Laboratory of Molecular Imaging, Southeast University between August 2006 and August 2007. MATERIALS： A total of 128 Wistar rats aged 24 hours were selected, and hippocampal neurons were harvested for primary cultures. METHODS： On day 7, primary hippocampal neuronal cultures were treated with Egb761 （0, 25, 50, 100, 150, and 200μg/mL） under hypoxic/hypoglycemic or hypoglycemic culture conditions for 12, 24, and 36 hours, respectively. Hippocampal neurons cultured in primary culture medium served as control. MAIN OUTCOME MEASURES： Cell viability was assayed using 3-（4,5-Dimethylthiazol-2-yl）- 2,5-diphenyltetrazolium bromide （MTT）; fluorescence detection of β -secretase activity was performed; Western Blot was used to measure β -secretase expression. RESULTS： Cell viability under hypoxic/hypoglycemic or hypoglycemic culture conditions was significantly less than control cells （P 〈 0.05）. Under hypoxic/hypoglycemic or hypoglycemic culture conditions, treatment with 25 μg/mL Egb761 did not alter cell viability. However, 〉 25 μg/mL Egb761 induced greater cell viability （P 〈 0.05）. No differences were observed between hypoxic/hypoglycemic or hypoglycemic cells （P 〉 0.05）. α -secretase activity was increased after 12 hours in hypoxic/hypoglycemic culture （P 〈 0.01）. There were no significant differences between the 12-, 24-, or 36-hour Egb761 groups and the hypoxic/hypoglycemic groups （P 〉 0.05）. β -secretase activity was greater after 12, 24, and 36 hours in hypoxic/hypoglycemic culture conditions, compared with control conditions （P 〈 0.05）. β-secretase activity was significantly decreased in neurons treated with Egb761 for 12, 24, or 36 hours, compared with the hypoxichaypoglycemic group （P 〈 0.05）. β -secretase protein expression was significantly up-regulated in neurons cultured in hypoxic/hypoglycemic conditions for 12, 24, or 36 hours, compared to control cells （P 〈 0.05）, and was decreased compared to neurons treated with Egb761 （P 〈 0.05）. CONCLUSION： β -secretase expression and activity in rat neonatal hippocampal neurons were influenced by hypoxic and hypoglycemic culture. Egb761 played a protective role in hippocampal neurons damaged by chronic hypoxic and hypoglycemic culture conditions, possibly through its effect on β -secretase expression and activity.

Improving cognitive impairment by Tongxinluo via inhibiting expression of beta-secretase 1/beta-amyloid peptide in experimental vascular dementia

BACKGROUND： Tongxinluo has been clinically proven to be effective in improving memory and cognitive function in patients with post-stroke vascular dementia. Is the mechanism related to the deposition of beta-amyloid peptide （A β ） in hippocampus？ OBJECTIVE： To observe the effect of Tongxinluo on cognitive impairment in a mouse model with vascular dementia and the changes of A β deposition and β -secretase 1 （BACE1） expression. DESIGN： Randomized controlled study. SETTING： State Key Laboratory of Pharmaceutical Biotechnology of Nanjing University and Affiliated Drum Tower Hospital of Nanjing University Medical School. MATERIALS： The experiment was carried out in the State Key Laboratory of Pharmaceutical Biotechnology of Nanjing University and Affiliated Drum Tower Hospital of Nanjing University Medical School from March 2006 to January 2007. A total of 36 healthy Kunming mice, 18 of each gender, were chosen. The study was conducted in accordance with the National Regulations of Experimental Animal Administration, and all animal experiments were approved by the Committee of Experimental Animal Administration of Nanjing University. Tongxinluo was provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd. METHODS： All mice were randomly divided into 6 groups, including naive control （n=6）, sham-operated control （n=6） and experimental groups treated with different doses of Tongxinluo （0.2, 0.4, and 0.6 g/kg/d; n=6 for each group） or vehicle （n=6）. Five groups were subjected to bilateral common carotid arteries （2-VO） occlusion to produce a vascular dementia model （no occlusion was performed in sham-operated group）. The mice in the Tongxinluo treatment groups were intragastricly administered daily with a Tongxinluo suspension （40 g/L in distilled water） at doses of 0.2, 0.4 or 0.6 g/kg/d from day 1 to day 30 post-surgery. The animals in vehicle, sham-operated and naive groups were administered an equal volume of distilled water. MAIN OUTCOME MEASURES： ①Escape latency time determined in all groups of mice before and after 2-VO occlusion by Morris water maze. ②Changes in BACEI mRNA expression in the hippocampi of mice among the six groups by RT-PCR assay, and BACEI and A β protein expression in the hippocampi of mice by Western blot. RESULTS： All 36 mice were involved in the final analysis.① No difference was detected in escape latency time to a hidden platform among all groups in water maze test before surgery （P 〉 0.05） At 30 days after 2-VO occlusion, the vehicle animals exhibited a significantly longer latency in finding the hidden platform compared to that of sham-operated and naive animals （P 〈 0.01）. The prolonged escape latency was significantly reduced by oral administration of 0.4 g or 0.6 kg/day （P 〈 0.01, P 〈 0.05）. BACEI mRNA and protein expression in vehicle animals were much higher than in sham-operated and naive animals （P 〈 0.01）. The ischemia-induced increases in BACE1 mRNA and protein level were attenuated by all three doses of Tongxinluo treatment （P 〈 0.01）, and the 0.4 g/kg/d treatment was the most effective. A β protein expression in vehicle animals after 2-VO occlusion were much higher than in sham-operated and naive animals （P 〈 0.01）. 2-VO occlusion-induced A β generation was significantly attenuated by all doses of Tongxinluo treatment, with the most effective dose being 0.4 g/kg/d （P 〈 0.01）. CONCLUSION： BACE1 mRNA levels and protein levels of BACEI and A β are reduced in the hippocampi of vascular dementia model mice by all three doses of Tongxinluo treatment, with the most effective dose being 0.4 g/kg/d. The results suggest that inhibition of post-ischemia BACEI expression and A β generation in brain might underlie Tongxinluo＇s effects in improving cognitive impairment.

γ-Secretase Inhibitor, DAPT Inhibits Self-renewal and Stemness Maintenance of Ovarian Cancer Stem-like Cells In Vitro

Objective: The Notch signaling pathway plays an important role in the stem cell signaling network and contributes to tumorigenesis. However, the functions of Notch signaling in ovarian cancer stem cells (OCSCs) are not well understood. We aimed to investigate the effects of Notch blockade on self-renewal and stemness maintenance of OCSCs. Methods: Ovarian cancer stem-like cells were enriched from ovarian cancer cell lines in serum-free medium. A γ-secretase inhibitor, (DAPT), was used to block Notch signaling. MTT assays were performed to assess self-renewal and proliferation inhibition, flow cytometry was performed to analyze cell surface marker and immunofluorescence, Western Blot and Real-time RT-PCR assays were performed to detect Oct4 and Sox2 protein and mRNA expression of the Ovarian cancer stem-like cells treated with DAPT. Results: Notch blockade markedly inhibits self-renewal and proliferation of ovarian cancer stem-like cells, significantly downregulates the expression of OCSCs-specific surface markers, and reduces protein and mRNA expression of Oct4 and Sox2 in OCSC-like cells. Conclusion: Our results suggest that Notch signaling is not only critical for the self-renewal and proliferation of OCSCs, but also for the stemness maintenance of OCSCs. The γ-secretase inhibitor is a promising treatment targeting OCSCs.

Pharmacological Assessment of <i>γ</i>-Secretase Activity from Rodent and Human Brain

γ-Secretase is involved in the final processing of the amyloid precursor protein into a heterogeneous pool of β-amyloid (Aβ) peptides. Current Alzheimer’s disease drug discovery efforts include targeting γ-secretase activity in brain to attenuate production of the neurotoxic Aβ species. The resulting pharmacology may be affected by species-specific differences in the γ-secretase core complex or its associated proteins. Therefore, we utilized partially purified γ-secretase membranes derived from the brains of different species, including human cortex, to quantitatively assess the de novo production of both Aβ42 and Aβ40 following treatment with known γ-secretase inhibitors and modulators. We determined that the inhibitory activity of a Notch-1 sparing γ-secretase inhibitor and the modulatory activity of two classes of γ-secretase modulators were equipotent at affecting the production of Aβ across rodent and human brain membrane preparations. Additionally, the observed modulator-specific Aβ profile in isolated brain membranes across species was similar to that observed in HeLa cell membranes, and the brain and CSF of guinea pigs following oral administration. By utilizing rapidly purified γ-secretase, we were able to probe and compare the complex pharmacology of γ-secretase in the brain across common rodent species and human cortex.

BACE1 inhibitors:A promising therapeutic approach for the management of Alzheimer’s disease

Alzheimer’s disease is a neurological disorder marked by the accumulation of amyloid beta(Aβ)aggregates,resulting from mutations in the amyloid precursor protein.The enzymeβ-secretase,also known asβ-site amyloid precursor protein cleaving enzyme 1(BACE1),plays a crucial role in generating Aβpeptides.With no targeted therapy available for Alzheimer’s disease,inhibiting BACE1 aspartic protease has emerged as a primary treatment target.Since 1999,compounds demonstrating potential binding to the BACE1 receptor have advanced to human trials.Structural optimization of synthetically derived compounds,coupled with computational approaches,has offered valuable insights for developing highly selective leads with drug-like properties.This review highlights pivotal studies on the design and development of BACE1 inhibitors as anti-Alzheimer’s disease agents.It summarizes computational methods employed in facilitating drug discovery for potential BACE1 inhibitors and provides an update on their clinical status,indicating future directions for novel BACE1 inhibitors.The promising clinical results of Elenbecestat(E-2609)catalyze the development of effective,selective BACE1 inhibitors in the future.

分泌性BACE1蛋白在昆虫细胞的表达

为了获得有活性的重组β-secretase并研究其功能,寻找特异性抑制剂,应用RT-PCR技术,从人胚胎脑组织特异性扩增并克隆了人BACE1编码基因的胞外片断(BACE1-454)。测序后与质粒pFastBac连接,得到含BACE1-454基因的重组质粒pFast-BACE1-454。将其转化到含有杆状病毒穿梭载体Bacmid的感受态细胞DH10Bac中进行转座重组,用琼脂糖凝胶电泳和PCR扩增对重组穿梭载体Bacmid-BACE1-454进行鉴定。将Bacmid-BACE1-454经脂质体介导转染Sf9细胞,收获病毒。用重组杆状病毒颗粒感染昆虫细胞,表达蛋白,免疫印迹证实所获蛋白产品具有特异的免疫反应性。

Multitarget therapeutic strategies for Alzheimer's disease

Neurodegenerative diseases such as Alzheimer's, Huntington's and Parkinson's diseases have multifaceted nature because of the different factors contributing to their progression. The complex nature of neurodegenerative diseases has developed a pressing need to design multitarget-directed ligands to address the complementary pathways involved in these diseases. The major enzyme targets for development of therapeutics for Alzheimer's disease are cholinesterase and β-secretase enzymes. In this review, we discuss recent advances in profiling single target inhibitors based on these enzymes to multitarget-directed ligands as potential therapeutics for this devastating disease. In addition, therapeutics based on iron chelation strategy are discussed as well.

Computational Assessment and Pharmacological Property Breakdown of Eight Patented and Candidate Drugs against Four Intended Targets in Alzheimer’s Disease

Alzheimer’s Disease (AD) is the most prevalent age-related dementia. AD can be caused by abnormal processing of amyloid precursor protein (APP) or by oxidative stress or may be due to the actions of kinases or the degeneration and loss of functions of neurons in the brain. Although various treatments have already gained success in the in vitro studies, however, till now not a single satisfactory drug has been proven that can cure this disease permanently till now. In this study, the best possible drug has been determined from a group of drug molecules using methods of molecular docking. Molecular docking is a computational approach which helps to determine the best molecule from a group of molecules which may bind with the highest affinity with the intended target by mimicking the original biological environment in a computer. The tested drug molecules in this experiment are the disease modifying agents, capable of inhibiting a particular protein involving in the AD pathway. Eight drug molecules (ligands)-memantine (-4.075 Kcal/mol), hymenialdisine (-8.079 Kcal/mol), tideglusib (-6.445 Kcal/mol), kenpaullone (-7.545 Kcal/mol), dihydrospiro[dibenzo[a,d][7]annulene-5,4’-imidazol] (-4.742 Kcal/mol), harmine (-7.57 Kcal/mol), harmol (-6.583 Kcal/mol) and 1-Methyl-4-Phenylpyridinium (-5.214 Kcal/mol), have been docked successfully against four targets (proteins)-N-Methyl-D-Aspartate Receptor (NMDAR), glycogen synthase kinase-3β (GSK-3β), beta-secretase (β-secretase) and dual specificity tyrosine (Y)-phosphorylation-regulated kinase 1A (DYR-K1A) in this experiment which are intended targets in current AD treatment approaches. Investigation of docking results, druglikeness properties and ADME/T testing results suggest that the best findings of this experiment are memantine, hymenialdisine, dihydrospiro[dibenzo[a,d][7]annulene-5,4’-imi- dazol] and harmol, that could be the best possible drugs for the treatment of AD.

γ-secretase inhibitor DAPT prevents neuronal death and memory impairment in sepsis associated encephalopathy in septic rats

Background Brain dysfunction is a frequent complication of sepsis,usually defined as sepsis-associated encephalopathy （SAE）.Although the Notch signaling pathway has been proven to be involved in both ischemia and neuronal proliferation,its role in SAE is still unknown.Here,the effect of the Notch signaling pathway involved γ-secretase inhibitor DAPT on SAE in septic rats was investigated in a cecal ligation and puncture （CLP） model.Methods Fifty-nine Sprague-Dawley rats were randomly divided into four groups,with the septic group receiving the CLP operation.Twenty-four hours after CLP or sham treatment,rats were sacrificed and their hippocampus was harvested for Western blot analysis.TNF-αexpression was determined using an enzyme-linked immunosorbent assay （ELISA） kit.Neuronal apoptosis was assessed by TUNEL staining,and neuronal cell death was detected by H＆E staining.Finally,a novel object recognition experiment was used to evaluate memory impairment.Results Our data showed that sepsis can increase the expression of hippocampal Notch receptor intracellular domain （NICD） and poly （adenosine diphosphate [ADP]-ribose） polymerase-1 （PARP-1）,as well as the inflammatory response,neuronal apoptosis,neuronal death,and memory dysfunction in rats.The γ-secretase inhibitor N-[N-（3,5-difluorophenacetyl）-1-alanyl]-S-phenylglycine t-butyl ester （DAPT） can significantly decrease the level of NICD and PARP-1,reduce hippocampal neuronal apoptosis and death,attenuate TNF-α release and rescue cognitive impairment caused by CLP.Conclusion The neuroprotective effect of DAPT on neuronal death and memory impairment in septic rats,which could be a new therapeutic approach for treating SAE in the future.

Notch1 downregulation combined with interleukin-24 inhibits invasion and migration of hepatocellular carcinoma cells

AIM: To confirm the anti-invasion and anti-migration effects of down-regulation of Notch1 combined with interleukin(IL)-24 in hepatocellular carcinoma(HCC) cells.METHODS: γ-secretase inhibitors(GSIs) were used to down-regulate Notch1.Hep G2 and SMMC7721 cells were seeded in 96-well plates and treated with GSI-I or/and IL-24 for 48 h.Cell viability was measured by MTT assay.The cellular and nuclear morphology was observed under a fluorescence microscope.To further verify the apoptotic phenotype,cell cultures were also analyzed by flow cytometry with Annexin V-FITC/propidium iodide staining.The expression of Notch1,SNAIL1,SNAIL2,E-cadherin,IL-24,XIAP and VEGF was detected by Western blot.The invasion and migration capacities of HCC cells were detected by wound healing assays.Notch1 and Snail were downregulated by RNA interference,and the target proteins were analyzed by Western blot.To investigate the mechanism of apoptosis,we analyzed Hep G2 cells treated with si Notch1 or si CON plus IL-24 or not for 48h by caspase-3/7 activity luminescent assay.RESULTS: GSI-I at a dose of 2.5 μmol/L for 24 h caused a reduction in cell viability of about 38% in Hep G2 cells.The addition of 50 ng/m L IL-24 in combination with 1 or 2.5 μmol/L GSI-I reduced cell viability of about 30% and 15%,respectively.Treatment with IL-24 alone did not induce any cytotoxic effect.In SMMC7721 cells with the addition of IL-24 to GSI-I(2.5 μmol/L),the reduction of cell viability was only about 25%.Following GSI-I/IL-24 combined treatment for 6 h,the apoptotic rate of Hep G2 cells was 47.2%,while no significant effect was observed in cells treated with the compounds employed separately.Decreased expression of Notch1 and its associated proteins SNAIL1 and SNAIL2 was detected in Hep G2 cells.Increased E-cadherin protein expression was noted in the presence of IL-24 and GSI-I.Furthermore,the increased GSI-I and IL-24 in Hep G2 cell was associated with downregulation of MMP-2,XIAP and VEGF.In the absence of treatment,Hep G2 cells could migrate into the scratched space in 24 h.With IL-24 or GSI-I treatment,the wound was still open after 24 h.And the distance of the wound closure strongly correlated with the concentrations of IL-24 and GSI-I.Treatment of Notch-1 silenced Hep G2 cells with 50 ng/m L IL-24 alone for 48 h induced cytotoxic effects very similar to those observed in non-silenced cells treated with GSI-I/IL-24 combination.Caspase-3/7 activity was increased in the presence of si Notch1 plus IL-24 treatment.CONCLUSION: Down-regulation of Notch1 by GSI-I or si RNA combined with IL-24 can sensitize apoptosis and decrease the invasion and migration capabilities of Hep G2 cells.