Lineweaver-Burk Plot Analysis Research Articles

The first bioactive (angiotensin-converting enzyme-inhibitory) peptide isolated from pearl matrix protein

In this research, we unveil the medical potential of pearls by identifying a novel bioactive peptide within them for the first time. The peptide, termed KKCHFWPFPW, emerges as a pioneering angiotensin I-converting enzyme (ACE) inhibitor, originating from the pearl matrix of Pinctada fucata. Employing quadrupole time-of-flight mass spectrometry, this peptide was meticulously selected and pinpointed. With a molecular weight of 1417.5 Da and a theoretical isoelectric point of 9.31, its inhibitory potency was demonstrated through a half-maximal inhibitory concentration (IC50) of 4.17 μM, established via high-performance liquid chromatography. The inhibition of ACE by this peptide was found to be competitive, as revealed by Lineweaver–Burk plot analysis, where an increase in peptide concentration correlated with an enhanced rate of ACE inhibition. To delve into the interaction between KKCHFWPFPW and ACE, molecular docking simulations were conducted using the Maestro 2022-1 Glide software, shedding light on the inhibitory mechanism. This investigation suggests that peptides derived from the P. martensii pearl matrix hold promise as a novel source for antihypertensive agents.

New amide and diterpene alkaloids with anticholinesterase activity from Delphinium cyphoplectrum roots.

The cholinergic hypothesis posits a robust correlation between the onset of Alzheimer's disease and a pronounced deficit in acetylcholine, a pivotal neurotransmitter crucial for the central cholinergic nervous system's function, pivotal for memory and learning. Diterpene alkaloids exhibit intricate and distinctive chemical structures that facilitate their passage through the blood-brain barrier. Moreover, their potent pharmacological attributes render them promising candidates for addressing central nervous system disorders. This investigation aims to scrutinize the alkaloidal composition of Delphinium cyphoplectrum (Ranunculaceae) roots, further exploring their anticholinesterase inhibitory activity and mode of inhibition. Innovative chromatography techniques were repetitively employed to purify the alkaloids. Acetylcholinesterase (AChE) inhibition assays were conducted using Ellman's tests. The mode of inhibition was meticulously characterized through Michaelis-Menten, and Lineweaver-Burk plots. Conducting molecular docking studies, we employed the AUTO DOCK 4.2 software package. Eight alkaloids were identified including five C19-diterpene alkaloids (6,14,16,18-tetramethoxy-1,7,8-trihydroxy-4-methylaconitane (1), 6,16,18-trimethoxy-1,7,8,14-tetrahydroxy-4-methylaconitane (2), 6,8,16,18-tetramethoxy-1,7,14-trihydroxy-4-methylaconitane (3), 6,14,16-trimethoxy-1,7,8,18-tetrahydroxy-4-methylaconitane (4), and 14-O-acetyl-8,16-dimethoxy-1,6,7,18-tetrahydroxy-4-methylaconitane (5)), an epoxy C18-diterpene alkaloid (6,8,16-trimethoxy-1,7,14-trihydroxy-3,4-epoxyaconitane (6)), a known (pyrrolidin-2-one (7) and an undescribed amide alkaloid (1-(2'-hydroxylethylamine)-3,5,5,-trimethyl-1,5-dihydro-2H-pyrrol-2-one (8). All diterpene alkaloids underwent assessment for acetylcholinesterase (AChE) inhibition assay and displayed noteworthy AChE activity, surpassing that of the reference drug (with IC50 values of 13.7, 21.8, 23.4, 28.2, 40.4, and 23.9 for compounds 1-6, respectively, in comparison to 98.4 for Rivastigmine). Analysis of Michaelis-Menten and Lineweaver-Burk plots represents an uncompetitive mode of inhibition for compound 1 on AChE. Notably, computational docking simulations indicated that all diterpene alkaloids were accommodated within the same enzymatic cleft as the reference ligand, and displaying superior free binding energy values (from - 10.32 to -8.59 Kcal.mol-1) in contrast to Rivastigmine (-6.31 Kcal.mol-1). The phytochemical analysis conducted on the roots of Delphinium cyphoplectrum yielded the identification of eight alkaloidal compounds including one C18-diterpene, five C19-diterpene, one pyrrolidine and one amide alkaloids. AChE inhibition assay and molecular simulations unveiled remarkable significant potency attributed to the C19-diterpene alkaloids by the order of 1 > 2 > 3,6 > 4 > 5. Presence of hydroxyl group on C-1, C-7, C-8, C-14, and C-18 increased the effect. The best in vitro activity was recorded for compound 1 able to bind to Asp72 in the narrow region of PAS, while interacting by pi-sigma with Phe330 at the hydrophobic region of the gorge involving the acyl and choline binding site. This observation underscores the substantial promise of this category of natural products in the realm of drug discovery for Alzheimer's Disease, offering a compelling avenue for further research and therapeutic development.

Piperazine-2-carboxylic acid derivatives as MTDLs anti-Alzheimer agents: Anticholinesterase activity, mechanistic aspect, and molecular modeling studies

Development of Multitarget-Directed Ligands (MTDLs) is a promising approach to combat the complex etiologies of Alzheimer's disease (AD). Herein we report the design, synthesis, and characterization of a new series of 1,4-bisbenzylpiperazine-2-carboxylic acid derivatives 3-5(a-g), 7a-f, 8a-s, and their piperazine-2-yl-1,3,4-oxadiazole analogs 6a-g. In vitro inhibitory effect against Electrophorus electricus acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) from Equine serum was evaluated using modified Ellman’s method, considering donepezil and tacrine as reference drugs. Lineweaver–Burk plot analysis of the results proved competitive inhibition of AChE and BChE with Ki values, in low micromolar range. The free carboxylic acid series 4a-g showed enhanced selectivity for AChE. Hence, 4c, 1,4-bis (4-chlorobenzyl)-piperazinyl-2-carboxylic acid), was the most active member of this series (Ki (AChE) = 10.18 ± 1.00 µM) with clear selectivity for AChE (SI ∼ 17.90). However, the hydroxamic acids 7a-f and carboxamides 8a-s congeners were more potent and selective inhibitors of BChE (SI ∼ 5.38 – 21862.5). Extraordinarily, 1,4-bis (2-chlorobenzyl)-piperazinyl-2-hydroxamic acid 7b showed promising inhibitory activity against BChE enzyme (Ki = 1.6 ± 0.08 nM, SI = 21862.5), that was significantly superior to that elicited by donepezil (Ki = 12.5 ± 2.6 µM) and tacrine (Ki = 17.3 ± 2.3 nM). Cytotoxicity assessment of 4c and 7b, on human neuroblastoma (SH-SY5Y) cell lines, revealed lower toxicity than staurosporine and was nearly comparable to that of donepezil. Molecular docking and molecular dynamics simulation afforded unblemished insights into the structure–activity relationships for AChE and BChE inhibition. The results showed stable binding with fair H-bonding, hydrophobic and/or ionic interactions to the catalytic and peripheral anionic sites of the enzymes. In silico predicted ADME and physicochemical properties of conjugates showed good CNS bioavailability and safety parameters. In this regard, compound (7b) might be considered as a promising inhibitor of BChE with an innovative donepezil-based anti-Alzheimer activity. Further assessments of the most potent AChE and BChE inhibitors as potential MTDLs anti-Alzheimer’s agents are under investigation with our research group and will be published later.

Antigout Activity of The Spatholobus littoralis Hassk. Extract Fractions Against Xanthine Oxidase: Its Metabolite Profile and Inhibition Kinetics

The Spatholobus littoralis Hassk. (S. littoralis Hassk.) is a native plant in Indonesia and has been widely used in traditional Dayaknese medicine for non-communicable degenerative diseases. One of these illnesses, known as gout, is caused by excessive uric acid in the blood, which is the catalytic byproduct of a xanthine oxidase (XO) enzyme. In this work, we investigated the inhibition kinetics of XO and identified bioactive compounds from the stem extract fractions of S. littoralis Hassk. using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Fractionation was carried out to obtain n-hexane, dichloromethane, and 1-butanol fractions from the water and 70% ethanol extracts. Fraction of 1-butanol from 70% ethanol and water extract displayed potent inhibitors of XO with IC50 value 116.91±3.51 and 137.15±5.00 mg/L, respectively. Lineweaver-Burk plot analysis showed that the 1-butanol fraction from the two extracts inhibited XO competitively. The 1-butanol fraction from the two extracts has been further identified as a bioactive fraction. The majority of the compounds in the two active fractions were phenolics. These findings revealed that the 1-butanol fraction from the two extracts is promising as an antigout treatment in the future.

Enzymatic properties and clinical associations of serum alpha-galactosidase A in Parkinson's disease.

Recent studies have revealed an association between Parkinson's disease (PD) and Fabry disease, a lysosomal storage disorder; however, the underlying mechanisms remain to be elucidated. This study aimed to investigate the enzymatic properties of serum alpha-galactosidase A (GLA) and compared them with the clinical parameters of PD. The study participants consisted of 66 sporadic PD patients and 52 controls. We measured serum GLA activity and calculated the apparent Michaelis constant (Km ) and maximal velocity (Vmax ) by Lineweaver-Burk plot analysis. Serum GLA protein concentration was measured by enzyme-linked immunosorbent assay. We examined the potential correlations between serum GLA activity and GLA protein concentration and clinical features and the plasma neurofilament light chain (NfL) level. Compared to controls, PD patients showed significantly lower serum GLA activity (P < 0.0001) and apparent Vmax (P = 0.0131), but no change in the apparent Km value. Serum GLA protein concentration was lower in the PD group (P = 0.0168) and was positively associated with GLA activity. Serum GLA activity and GLA protein concentration in the PD group showed a negative correlation with age. Additionally, serum GLA activity was negatively correlated with the motor severity score and the level of plasma NfL, and was positively correlated with the score of frontal assessment battery. This study highlights that the lower serum GLA activity in PD is the result of a quantitative decrement of GLA protein in the serum and that it may serve as a biomarker of disease severity.

The Molecular Docking and Inhibition Kinetics of Angiotensin I-Converting Enzyme Inhibitory Peptides Derived from Soft-Shelled Turtle Yolk

The soft-shelled turtle yolk (SSTY) protein hydrolysate contains a potential source of bioactive peptides. Our previous study found that five SSTY peptides (WLQL, LPSW, LPLF, VPGLAL and LVGLPL) showed moderate to high dipeptidyl peptidase IV (DPP-IV) inhibitory activities. This study further investigated their angiotensin-I-converting enzyme (ACE) inhibitory activity. Consequently, WLQL was identified as the most potent ACE inhibitory peptide with a remarkably low IC50 value (16.87 ± 0.54 µM). The Lineweaver–Burk plot analysis was performed for the characterization of the peptide’s inhibition mode and the inhibition kinetics was rationalized using the molecular docking simulation. The result revealed that WLQL would dock into the S1 pockets of ACE, while LPSW interacted with ACE’s secondary binding site. Further evaluation of the peptides’ stability against ACE involved a pre-incubation experiment. After 3 h of pre-incubation with ACE, the four peptides were hydrolyzed into smaller fragments with varying degrees, suggesting that they are substrate-type inhibitors. In contrast, LVGLPL can tolerate hydrolysis by ACE and act as a true inhibitor.

Cholinesterase inhibitory activities of neuroprotective fraction derived from red alga Gracilaria manilaensis

Anti-cholinesterase (ChE)s are commonly prescribed as the symptomatic treatment of Alzheimer’s disease. They are applied to prevent the breakdown of neurotransmitter acetylcholine (ACh) that bind to muscarinic and nicotinic receptors in the synaptic cleft. Seaweeds are one of the richest sources of bioactive compounds for both nutraceuticals and pharmacognosy applications. This study aimed to determine the anti-ChEs activity of Gracilaria manilaensis, one of the red seaweeds notables for its economic importance as food and raw materials for agar production. Methanol extracts (GMM) of G. manilaensis were prepared through maceration, and further purified with column chromatography into a semi-pure fraction. Ellman assay was carried out to determine the anti-acetylcholinesterase (AChE) and anti-butyrylcholinesterase (BuChE) activities of extracts and fractions. Lineweaver-Burk plot analysis was carried out to determine the inhibition kinetic of potent extract and fraction. Major compound(s) from the most potent fraction was determined by liquid chromatography-mass spectrometry (LCMS). GMM and fraction G (GMMG) showed significant inhibitory activity AChE with EC50 of 2.6 mg/mL and 2.3 mg/mL respectively. GMM and GMMG exhibit mixed-inhibition and uncompetitive inhibition respectively against AChE. GMMG possesses neuroprotective compounds such as cynerine A, graveolinine, militarinone A, eplerenone and curumenol. These findings showed a promising insight of G. manilaensis to be served as a nutraceutical for neuronal health care in the future.

Proanthocyanidins as the main pancreatic lipase inhibitors in chokeberry fruits.

Pancreatic lipase inhibitors are recognized as important in strategies for the management of overweight and obesity. The phytocompounds in chokeberry fruit show multidirectional pro-health effects, including anti-obesity activity. The aims of this study were to fractionate and identify the phenolic compounds of chokeberry fruit phenolic-rich extract that are active as pancreatic lipase inhibitors. Phenolic compounds were fractionated using Sephadex LH-20 resin, followed by polyphenol profile analysis using chromatographic and spectrophotometric methods, while pancreatic inhibitory activity was determined using 4-methylumbelliferyl oleate and emulsified triolein as enzyme substrates. Among the six fractions isolated from extract, two fractions rich in highly polymerized proanthocyanidins showed the greatest ability to inhibit pancreatic lipase activity. In comparison, fractions containing mainly low-molecular-weight phenolic compounds, such as phenolic acids, flavonols and anthocyanins, were 11-64 times less active. The most active fraction showed a mixed mode of pancreatic lipase inhibition, as determined by Lineweaver-Burk plot analysis, and exhibited a cumulative effect with orlistat. This study shows that black chokeberry polyphenols, particularly highly polymerized procyanidins, can effectively inhibit pancreatic lipase activity determined by in vitro methods.

Phenolic fraction concentrate of Phoenix dactylifera L. seeds: A promising antioxidant and glucose regulator

Context: The beneficial effects of natural polyphenolic compounds are increasingly emerging as powerful antioxidants and digestive boosters. Phoenix dactylifera seeds are recently identified as a healthy functional food with rich amounts of phenolic compounds. Aims: To analyzing the phenolic profile obtained from Moroccan variety of date seeds and evaluating the inhibitory capacity against digestive enzymes and the antioxidant potential of phenolic fraction concentrate (PFC) of date seeds as a promising antihyperglycemic agent. Methods: Lineweaver–Burk plot analysis was performed to evaluate PFC inhibitory effect against digestive enzyme activity in the presence of different doses of a substrate. Wistar rats were recruited to determine the acute and sub-acute oral toxicity of PFC. Finally, the optimal dose of PFC was selected and its effects on metabolic, hematological and biochemical parameters were determined. Results: High-performance liquid chromatography (HPLC) analysis revealed that PFC contains abundant amounts of phenolic acid: Caffeic acid, chlorogenic acid, 3,4-dihydroxybenzoic acid, p-hydroxybenzoic acid, and 3-hydroxybenzoic acid. PFC showed powerful antioxidant activity as determined by scavenging and reducing assays. PFC significantly inhibited alpha-amylase and alpha-glucosidase enzymes by 87.3% and 78.7%, respectively, via mixed manipulation of Km and Vmax. Acute toxicity examinations revealed PFC to be practically non-toxic with LD50 &gt; 5000mg PFC/kg for both sexes of rats. The no-observed-adverse-effect-level (NOAEL) and lowest observed adverse effect level (LOAEL) for oral administration of PFC were 1000 and 2500 mg/kg for both sexes. Finally, the sub-acute study showed that a PFC dose of 50 mg/kg did not cause any changes in metabolic, biochemical and hematological parameters and was considered to be the optimal healthy dose for rats. Consequently, it induces improvement in glucose concentration in rats. Conclusions: The rational use of phenolic date seeds fraction presents powerful antioxidant and blood glucose regulating nutraceutical that could aid in diabetes mellitus management.

Synthesis and biological evaluation of 2-(4-alkoxy-3-cyano)phenylpyrimidine derivatives with 4-amino or 4-hydroxy as a pharmacophore element binding with xanthine oxidase active site

Xanthine oxidase is the rate-limiting enzyme critical for the synthesis of uric acid, and therefore xanthine oxidase inhibitors are considered as one of the promising therapies for hyperuricemia and gout. In our previous study, series of 2-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acids and 2-(4-alkoxy-3-cyano)phenyl-6-imino-1,6-dihydropyrimidine-5-carboxylic acids were synthesized that presented excellent in vitro xanthine oxidase inhibitory potency. Interestingly, molecular docking studies revealed that the interaction behavior of these compounds with xanthine oxidase was changed after the conversion from a hydroxy group to amine group. To further investigate the structure–activity relationships of these pyrimidine-containing xanthine oxidase inhibitors and explore the contribution of amino or hydroxy group on xanthine oxidase inhibitory potency, several 2-phenylpyrimidine derivatives with amino or hydroxy functional group were designed and synthesized. Thereafter, the structure–activity research and molecular modeling study proved that hydroxy and amino groups could be used as pharmacophore elements for the design of 2-phenylpyrimidines xanthine oxidase inhibitors. Particularly, the optimized compound, 2-(3-cyano-4-isopentoxy)phenylpyrimidine-4-ol, emerged the strongest xanthine oxidase inhibitor potency, with an IC50 value of 0.046 µM, which was approximately 120-fold more potent than that of allopurinol (IC50 = 5.462 µM). Additionally, Lineweaver-Burk plot analysis revealed that the optimized compound acted as a mixed-type inhibitor. Furthermore, the in vivo hypouricemic effect of the optimized compound was investigated in a hyperuricemia rat model induced by potassium oxonate, and the results showed that the optimized compound could effectively reduce serum uric acid levels at an oral dose of 30 mg/kg.

Anti-Aging Potential of Extracts from Washingtonia filifera Seeds.

The aim of this study was to test the inhibitory effect of fruit extracts from Washingtonia filifera on skin aging-related enzymes. The pulp extracts did not exert a significant enzyme inhibition while seed extracts from W. filifera exhibit anti-elastase, anti-collagenase, and anti-tyrosinase activities. Tyrosinase was mildly inhibited while a stronger effect was observed with respect to elastase and collagenase inhibition. Alcoholic extracts provided better results than aqueous extracts. Among them, methanol extracts showed the prominent enzyme inhibitory activities being IC50 value for elastase and collagenase comparable and even better than the reference compound. The inhibition mode of the most active extracts was investigated by Lineweaver-Burk plot analysis. Seed extracts from W. filifera were also investigated for their photo-protective effect by Mansur equation and the antioxidant activity of W. filifera extract was evaluated in oxidative-stressed cells. To evaluate the safety of the extract, the effect on cell viability of human keratinocytes cells was analyzed. Methanol extract presented the best photo-protective effect and exerted an antioxidant activity in a cellular system with no cytotoxic effect. The overall results demonstrate that W. filifera extracts are promising sources of bioactive compounds that could be used in cosmetic and pharmaceutical preparation.

Washingtonia filifera seed extracts inhibit the islet amyloid polypeptide fibrils formations and α-amylase and α-glucosidase activity

Washingtonia filifera seeds have revealed to possess antioxidant properties, butyrylcholinesterase and xanthine oxidase inhibition activities. The literature has indicated a relationship between Alzheimer’s disease (AD) and type-2 diabetes (T2D). Keeping this in mind, we have now evaluated the inhibitory properties of W. filifera seed extracts on α-amylase, α-glucosidase enzyme activity and the Islet Amyloid Polypeptide (IAPP) fibrils formation. Three extracts from seeds of W. filifera were evaluated for their enzyme inhibitory effect and IC50 values were calculated for all the extracts. The inhibition mode was investigated by Lineweaver-Burk plot analysis and the inhibition of IAPP aggregate formation was monitored. W. filifera methanol seed extract appears as the most potent inhibitor of α-amylase, α-glucosidase, and for the IAPP fibril formation. Current findings indicate new potential of this extract that could be used for the identification or development of novel potential agents for T2D and AD.

Comparison of Antioxidant and α-Glucosidase Inhibitory Activities in Different Cultivars of Five Mango (Mangifera Indica L.) Leaf Extracts

The objectives of study were to evaluate and compare the antioxidant, total phenolic, total flavonoid, mangiferin content and antidiabetic activities of five young mango cultivars leaf extract, namely, ‘Apple’, ‘Nam Dok Mai’, ‘Bao’, ‘OkRong’ and ‘Kiew Savoey’. Antioxidant effect was investigated by DPPH, ABTS radical scavenging activity, and ferric reducing power (FRAP) assays. Inhibitory on α-glucosidase activity and type of enzyme inhibition were evaluated by using Lineweaver Burk plot analysis. Mangiferin, major active compound, was quantified by HPTLC method. Furthermore, the hypoglycemic effect was determined using streptozotocin (STZ) –nicotinamide (NA) -induced type 2 diabetic mice. Young mango cv. ‘Apple’ leaf extract demonstrated the strongest antioxidant activity in all assays. Moreover, it contains highest amounts of total phenolic and mangiferin to the values of 311 mg GAE/g extract and 197 mg/g extract, respectively. It possessed potent α-glucosidase inhibitory activity with IC50 value of 0.50 µg/mL. Lineweaver-Burk plot analysis demonstrated a non-competitive inhibition of αglucosidase activity with the inhibition constant (Ki) of 2.98 µg/mL. Coadministration of young mango cv. ‘Apple’ leaf extract at dose of 1,000 mg/kg significantly reduced the total blood glucose level by 13.43% in STZ-NA-induced type 2 diabetic mice when compared with control diabetic mice in oral glucose tolerance test (OGTT) model. Inhibition of glucose absorption may be one of the possible mechanism of its hypoglycemic effect. In conclusion, young mango cv. ‘Apple’ leaf extract possesses the strongest antioxidant and antidiabetic activities which has a potential to develop as nutraceutical products.

Bioactive flavonoids from Rubus corchorifolius inhibit α-glucosidase and α-amylase to improve postprandial hyperglycemia

This research established an optimized method for the extraction and enrichment of flavonoids from R. corchorifolius fruit. Under the optimized extraction conditions, 12 flavonoids (1–12) were isolated, of which six (2–4, 9–10, 12) were obtained from R. corchorifolius for the first time. Compound 4 showed significant α-glucosidase (4.96 μM) and α-amylase (8.04 μM) inhibitory effects. Molecular modeling revealed that compound 4 exhibits strong binding with the active sites of α-glucosidase and α-amylase. Lineweaver-Burk plot analysis and surface plasmon resonance revealed the possible dynamic binding mechanism of the flavonoids with α-glucosidase and α-amylase. The enriched flavonoids and compound 4 showed significant hypoglycemic effects in mice administered a high dose of glucose. In this study, a variety of flavonoids with hypoglycemic activity were found for the first time, revealing the rich chemical composition of R. corchorifolius fruit and highlighting the potential value of R. corchorifolius fruit flavonoids as dietary supplements.

Synthesis, α-glucosidase inhibition and in silico studies of some 4-(5-fluoro-2-substituted-1H-benzimidazol-6-yl)morpholine derivatives

In this study, a new series of 4-(5-fluoro-2-substituted-1H-benzimidazol-6-yl)morpholine derivatives has been synthesized and screened for their α-glucosidase inhibitory potential. All molecules showed a considerable α-glucosidase inhibitory potential with IC50 values ranging from 20.46 ± 0.21 to 0.18 ± 0.01 µg/mL when compared with the acarbose (IC50 = 8.16 ± 0.12 µg/mL) as the standard. Compound 4 k having methoxy group on phenyl ring had the highest inhibitory effect with IC50 = 0.18 ± 0.01 µg/mL value among the examined compounds. Electron-donating groups such as methyl and methoxy on the phenyl ring played an important role in the inhibition. Also, the Lineweaver-Burk plots analysis displayed that the inhibition type of 4k was the competitive mode like acarbose as standard. In silico studies were also performed to explore the binding interaction of the most active compound.

Substrate-Specific Activation of α-Secretase by 7-Deoxy-Trans-Dihydronarciclasine Increases Non-Amyloidogenic Processing of β-Amyloid Protein Precursor.

Accumulation of β-amyloid (Aβ) in the brain has been implicated in the pathology of Alzheimer’s disease (AD). Aβ is produced from the Aβ precursor protein (APP) through the amyloidogenic pathway by β-, and γ-secretase. Alternatively, APP can be cleaved by α-, and γ-secretase, precluding the production of Aβ. Thus, stimulating α-secretase mediated APP processing is considered a therapeutic option not only for decreasing Aβ production but for increasing neuroprotective sAPPα. We have previously reported that 7-deoxy-trans-dihydronarciclasine (E144), the active component of Lycoris chejuensis, decreases Aβ production by attenuating APP level, and retarding APP maturation. It can also improve cognitive function in the AD model mouse. In this study, we further analyzed the activating effect of E144 on α-secretase. Treatment of E144 increased sAPPα, but decreased β-secretase products from HeLa cells stably transfected with APP. E144 directly activated ADAM10 and ADAM17 in a substrate-specific manner both in cell-based and in cell-free assays. The Lineweaver–Burk plot analysis revealed that E144 enhanced the affinities of A Disintegrin and Metalloproteinases (ADAMs) towards the substrate. Consistent with this result, immunoprecipitation analysis showed that interactions of APP with ADAM10 and ADAM17 were increased by E144. Our results indicate that E144 might be a novel agent for AD treatment as a substrate-specific activator of α-secretase.

Preparation and Irreversible Inhibition Mechanism Insight into a Recombinant Kunitz Trypsin Inhibitor from Glycine max L. Seeds

Soybean Kunitz trypsin inhibitor (SKTI), extracted from soybean (Glycine max L.) seeds, possesses insect resistance and anti-tumor properties. But its specific mechanisms of action are not yet known. This article reports an efficient method to produce recombinant SKTI (rSKTI) in Escherichia coli, reveals some biochemical properties of rSKTI, and discusses the inhibition mechanism of SKTI. The rSKTI was expressed as inclusion body in E. coli BL21 (DE3). After refolding, the active rSKTI was obtained and was further purified with anion-exchange chromatography (DEAE-FF) efficiently. There were similar biochemical properties between SKTI and rSKTI. The optimum pH and the optimum temperature were pH 8.0 and 35 °C, respectively, being stable during pH 7.0–11.0 and below 37 °C. The activity against trypsin was inhibited by Co2+, Mn2+, Fe3+, Al3+, and epoxy chloropropane. Inhibition kinetic assay of SKTI against trypsin as Lineweaver-Burk plots analysis both showed an unchanged Km and a decreased Vmax with N-benzoyl-l-arginine ethyl ester (BAEE) as substrate. Molecular modeling showed Arg63 of SKTI (active residue of SKTI) that interacts with four residues of trypsin, including three catalytic site (His57, Asp102, and Ser195) and one binding site (Asp189), forming five interactions. These provide reference for understanding the inhibition mechanism of such kind of Kunitz trypsin inhibitors.

Selective acetylcholinesterase inhibitors derived from muscle relaxant dantrolene

Dantrolene, the only therapeutic agent for malignant hyperthermia, is known to have not only a muscle relaxant effect, but also a neuroprotective effect and Alzheimer's disease improving effect. Recently, it has been reported that dantrolene has a weak inhibitory effect on acetylcholinesterase (AChE), which is a therapeutic drug target for Alzheimer's disease. Thus, we focused on developing of AChE inhibitors with benzylpiperidine/piperazine moieties that are based on the dantrolene skeleton. Several derivatives showed an inhibitory activity. Among them, ortho-nitro derivative 8c showed the most potent inhibitory activity with the IC50 value of 34.2 nM. Furthermore, Lineweaver-Burk plot analysis indicated that 8c is AChE-selective inhibitor, which shows only a weak inhibitory effect on butyrylcholinesterase (BuChE) and a non-competitive inhibition.

Design, synthesis and biological evaluation of 2-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acid derivatives as novel xanthine oxidase inhibitors

In our previous study, we reported a series of 1-hydroxy-2-phenyl-1H-imidazole-5-carboxylic acid derivatives that presented excellent in vitro xanthine oxidase (XO) inhibitory potency. To further investigate the structure-activity relationships of these compounds, the imidazole ring was transformed to a pyrimidine ring to design 2-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acids (8a-8j), 2-(4-alkoxy-3-cyano)phenyl-4-methyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acids (9c, 9e, 9j, 9l) and 2-(4-alkoxy-3-cyano)phenyl-6-imino-1,6-dihydropyrimidine-5-carboxylic acids (10c, 10e, 10j, 10l). These compounds exhibited remarkable in vitro XO inhibitory potency with IC50 values ranging from 0.0181 μM to 0.5677 μM. Specifically, compounds 10c and 10e, with IC50 values of 0.0240 μM and 0.0181 μM, respectively, emerged as the most potent XO inhibitors, and their potencies were comparable to that of febuxostat. Structure-activity relationship analysis revealed that the methyl group at 4-position of pyrimidine ring could damage the potency, and the XO inhibitory potency was maintained when carbonyl group was changed to an imino group. Lineweaver-Burk plot analysis revealed that the representative compound 10c acted as a mixed-type inhibitor. A potassium oxonate induced hyperuricemia model in rats was chosen to further confirm the hypouricemic effect of compound 10c, and the results showed that compound 10c (5 mg/kg) was able to significantly lower the serum uric acid level. Furthermore, in acute oral toxicity study, no sign of toxicity was observed when the mice were administered with a single 2000 mg/kg oral dose of compound 10c. These results suggested that compound 10c was a potent and promising uric acid-lowing agent for the treatment of hyperuricemia.

Gibberellin A&lt;sub&gt;3&lt;/sub&gt; mediated decreased transcriptional rate, mRNA stability and non-competitive inhibition of DNA methyltransferases in tobacco

The present work aims to examine the underlying molecular mechanism(s) for the reduced transcriptions and activities of DNA methyltransferases (NtDNMTs) by gibberellin A3 (GA3) in tobacco (Nicotiana tabacum L). Nuclear run-on and cordycepin chase assays demonstrated a significant reduction in the transcriptional rate and transcript half-lives of NtMET1 and NtCMT3 by GA3. Lineweaver-Burk plot analysis revealed a non-competitive mode of inhibition of NtDNMTs activity by GA3. Taken together, both in vivo and in vitro studies clearly demonstrated the altered transcriptional and post-transcriptional regulation as a mechanism of inhibition in expression and activity of NtDNMTs by GA3.