Identification of potential lead compounds against BACE1 through in-silico screening of phytochemicals of Medhya rasayana plants for Alzheimer's disease management.

Alzheimer's disease is a progressive and irreversible neurodegenerative disease that accounts for 70-80% of dementia in the elderly. According to recent clinical data, the incidence of the disease is exponentially increasing with age. Beta-site amyloid precursor protein cleaving enzyme1 (BACE1) is an important molecule involved in the pathogenesis of Alzheimer's disease due to its early role in the amyloid cascade. Cleavage of amyloid precursor protein by BACE1 is the rate-limiting step leading to the production and aggregation of amyloid-beta plaques. A number of natural products are being identified as non-competitive BACE1 inhibitors. In Ayurveda, Medhya rasayana is a group of medicinal herbs, specifically used for managing neurological disorders and is known to be effective in improving cognitivity and intellect. This study aimed to analyze the pharmacological activity of bio-active compounds in Medhya rasayana plants against BACE1, employing structure-based docking approach. 11 compounds out of 876 were identified as potential hits, based on docking scores, binding energies, and interactions with the critical residues of BACE1. Possible neurological activities of these compounds were predicted using PASS server. Out of the 11 compounds screened, two compounds, 'Convolidine' from the plant Convolvulus pleuricaulis Choisy and 'N-(4-hydroxybutyl) phthalimide' from Glycyrrhiza glabra satisfied the pharmacological parameters of Lipinski rule of filtering and ADMET prediction. The binding stability of these compounds against BACE1 was confirmed by molecular dynamic simulation and post dynamic MM/GBSA calculations. Detailed analysis of the interaction with the critical amino acids in the active site revealed the possible inhibitory potential of these compounds of medicinal plant origin against BACE1.

Artichoke cv. Francés flower extract as a rennet substitute: effect on textural, microstructural, microbiological, antioxidant properties, and sensory acceptance of miniature cheeses.

BACKGROUND: The most common milk-clotting enzymes in the cheese industry are recombinant chymosins. Food naturalness is a factor underpinning consumers' food choice. For consumers who avoid food with ingredients from genetically modified organisms (GMOs), the use of vegetable-based rennet substitute in the cheese formulation may be a suitable solution. Artichokes that deviate from optimal products, when allowed to bloom due to flower protease composition, are excellent as raw material for vegetable rennet preparation. As enzymatic milk clotting exerts a significant impact on the characteristics of the final product, this product should be studied carefully. RESULTS: Mature flowers from unharvested artichokes (Cynara scolymus cv. Francés) that did not meet aesthetic standards for commercialization were collected and used to prepare a flower extract. This extract, as a coagulant preparation, enabled the manufacture of cheeses with distinctive characteristics compared with cheeses prepared with chymosin. Rennet substitution did not affect the actual yield but led to significant changes in dry matter yield, humidity, water activity, protein content, and color, and conferred antioxidant activity to the cheeses. The rennet substitution promoted significant modifications in springiness, and in the microstructure of the cheese, with a more porous protein matrix and an increment in the size of the fat globules. Both formulations showed a similar microbiota evolution pattern with excellent microbiological quality and good sensory acceptance. CONCLUSIONS: The rennet substitute studied here produced a cheese adapted to specific market segments that demand more natural and healthier products made with a commitment to the environment but well accepted by a general cheese consumer. © 2020 Society of Chemical Industry.

Generative Adversarial Networks (GANs) Based Synthetic Sampling for Predictive Modeling.

In the present report we evaluate the possible utility of the Generative Adversarial Networks (GANs) in mapping the chemical structural space for molecular property profiles, with the goal of subsequently yielding synthetic (artificial) samples for ligand-based molecular modeling. Two case studies are considered: BACE-1 (ß-Secretase 1) and DENV (Dengue Virus) inhibitory activities, with the former focused on data populating and the latter on data balancing tasks. We train GANs using subsamples extracted from datasets for each bioactivity endpoint, and apply the trained networks in generating synthetic examples from the respective bioactivity chemical spaces. Original and synthetic samples are pooled together and employed to build BACE-1 and DENV inhibitory activity classifiers and their performance evaluated over tenfold external validation sets. In both case studies, the obtained classifiers demonstrate satisfactory predictivity with the former yielding accuracy (ACC) and Mathew's correlation coefficient (MCC) values of 0.80 and 0.59, while the latter produces balanced accuracy(BACC) and MCC values of 0.81 and 0.70, respectively. Moreover, the statistics of these classifiers are compared with those of other models in the literature demonstrating comparable to better performance. These results suggest that GANs may be useful in mapping the chemical space for molecular property profiles of interest, and thus allow for the extraction of synthetic examples for computational modeling.

F2X-Universal and F2X-Entry: Structurally Diverse Compound Libraries for Crystallographic Fragment Screening.

Crystallographic fragment screening (CFS) provides excellent starting points for projects concerned with drug discovery or biochemical tool compound development. One of the fundamental prerequisites for effective CFS is the availability of a versatile fragment library. Here, we report on the assembly of the 1,103-compound F2X-Universal Library and its 96-compound sub-selection, the F2X-Entry Screen. Both represent the available fragment chemistry and are highly diverse in terms of their 3D-pharmacophore variations. Validation of the F2X-Entry Screen in CFS campaigns using endothiapepsin and the Aar2/RNaseH complex yielded hit rates of 30% and 21%, respectively, and revealed versatile binding sites. Dry presentation of the libraries allows CFS campaigns to be carried out with or without the co-solvent DMSO present. Most of the hits in our validation campaigns could be reproduced also in the absence of DMSO. Consequently, CFS can be carried out more efficiently and for a wider range of conditions and targets.

Identification of embelin, a 3-undecyl-1,4-benzoquinone from Embelia ribes as a multitargeted anti-Alzheimer agent.

Beta-secreatse (BACE-1) and cholinesterases are clinically validated targets of Alzheimer's disease (AD), for which natural products have provided immense contribution. The multifaceted nature of AD signifies the need of multitargeted agents to tackle this disease. In the search of new natural products as dual BACE-1/cholinesterase inhibitors, a library of pure natural products was screened for inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and BACE-1. The screening efforts have identified 1,4-benzoquinone "embelin," a natural product derived from Embelia ribes displaying inhibition of all three enzymes, with IC50 values of 2.5, 5.4, and 2.1 µM, respectively. This screen has also identified isoquinoline alkaloids papaverine and L-tetrahydropalmatine as AChE inhibitors. Kinetic study has shown that embelin inhibits EeAChE and EqBChE with ki values of 4.59 and 0.57 µM, in an uncompetitive and noncompetitive manner, respectively. The interactions of embelin with allosteric peripheral anionic site of cholinesterases, has further supported the results of kinetic study. Embelin has also enhanced the activity of P-gp in LS-180 cells, the efflux pump which is involved in the clearance of amyloid-ß from AD brain. Further, the cell viability study in neuronal cell line has indicated the excellent therapeutic window of embelin. These results are indicative of the fact that embelin is a multitargeted agent playing role in stopping the formation of amyloid-ß oligomers (via inhibition of BACE-1), improves cholinergic-transmission (via inhibition of AChE/BChE) and increases amyloid-ß clearance (via P-gp induction).

Bioactive polycyclic polyprenylated acylphloroglucinols from Hypericum perforatum.

Fifteen new polycyclic polyprenylated acylphloroglucinols (PPAPs), hyperforatones A-O (1-15), along with 3 structurally related analogues (16-18), were isolated from the stems and leaves of Hypericum perforatum. Their structures and absolute configurations were established by a combination of NMR spectroscopic analyses, experimental and calculated electronic circular dichroism (ECD), modified Mosher's methods, Rh2(OCOCF3)4- and [Mo2(OAc)4]-induced ECD, X-ray crystallography, and the assistance of quantum chemical predictions (QCP) of 13C NMR chemical shifts. Compound 5 was found to be the first PPAP decorated by a rare 2,2,4,4,5-(pentamethyltetrahydrofuran-3-yl)methanol moiety and an oxepane ring. Furthermore, the isolates were screened for their acetylcholinesterase (AChE) and ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitory activities. Compounds 5, 10, 11, and 15 showed desirable AChE inhibitory activities (IC50 6.9-9.2 µM) and simultaneously inhibited BACE1 (at a concentration of 5 µM) with inhibition rates of 50.3%, 34.3%, 47.2%, and 34.6%, respectively. Interestingly, compound 5 showed the most balanced inhibitory activities against both AChE and BACE1 of all the tested compounds, which means that 5 could serve as the first valuable dual-targeted PPAP for the treatment of Alzheimer's disease. Preliminary molecular docking studies of 5 with BACE1 and AChE were also performed.

Heterologous expression and characterization of the aspartic endoprotease Pep4um from Ustilago maydis, a homolog of the human Chatepsin D, an important breast cancer therapeutic target.

The pep4um gene (um04926) of Ustilago maydis encodes a protein related to either vacuolar or lysosomal aspartic proteases. Bioinformatic analysis of the Pep4um protein revealed that it is a soluble protein with a signal peptide suggesting that it likely passes through the secretory pathway, and it has two probable self-activation sites, which are similar to those in Saccharomyces cerevisiae PrA. Moreover, the active site of the Pep4um has the two characteristic aspartic acid residues of aspartyl proteases. The pep4um gene was cloned, expressed in Pichia pastoris and a 54 kDa recombinant protein was observed. Pep4um-rec was confirmed to be an aspartic protease by specifically inhibiting its enzymatic activity with pepstatin A. Pep4um-rec enzymatic activity on acidic hemoglobin was optimal at pH 4.0 and at 40 °C. To the best of our knowledge this is the first report about the heterologous expression of an aspartic protease from a basidiomycete. An in-depth in silico analysis suggests that Pep4um is homolog of the human cathepsin D protein. Thus, the Pep4um-rec protein may be used to test inhibitors of human cathepsin D, an important breast cancer therapeutic target.

Deciphering the mechanism of potent peptidomimetic inhibitors targeting plasmepsins - biochemical and structural insights.

Malaria is a deadly disease killing worldwide hundreds of thousands people each year and the responsible parasite has acquired resistance to the available drug combinations. The four vacuolar plasmepsins (PMs) in Plasmodium falciparum involved in hemoglobin (Hb) catabolism represent promising targets to combat drug resistance. High antimalarial activities can be achieved by developing a single drug that would simultaneously target all the vacuolar PMs. We have demonstrated for the first time the use of soluble recombinant plasmepsin II (PMII) for structure-guided drug discovery with KNI inhibitors. Compounds used in this study (KNI-10742, 10743, 10395, 10333, and 10343) exhibit nanomolar inhibition against PMII and are also effective in blocking the activities of PMI and PMIV with the low nanomolar Ki values. The high-resolution crystal structures of PMII-KNI inhibitor complexes reveal interesting features modulating their differential potency. Important individual characteristics of the inhibitors and their importance for potency have been established. The alkylamino analog, KNI-10743, shows intrinsic flexibility at the P2 position that potentiates its interactions with Asp132, Leu133, and Ser134. The phenylacetyl tripeptides, KNI-10333 and KNI-10343, accommodate different ρ-substituents at the P3 phenylacetyl ring that determine the orientation of the ring, thus creating novel hydrogen-bonding contacts. KNI-10743 and KNI-10333 possess significant antimalarial activity, block Hb degradation inside the food vacuole, and show no cytotoxicity on human cells; thus, they can be considered as promising candidates for further optimization. Based on our structural data, novel KNI derivatives with improved antimalarial activity could be designed for potential clinical use. DATABASE: Structural data are available in the PDB under the accession numbers 5YIE, 5YIB, 5YID, 5YIC, and 5YIA.

Selected Cardoon (Cynara cardunculus L.) Genotypes Suitable for PDO Cheeses in Mediterranean Regions.

Cardoon flower extract is a traditional and exclusive rennet used for some PDO cheeses in several Mediterranean regions, due to its extremely high concentration in cardosins. In this preliminary study, six individual cardoon genotypes (1M - 6M) were selected because they revealed a wide and consistent diversity of total and specific cardosin concentrations in flowers. During three growing seasons, the stability of 12 biochemical characteristics of flower extracts and 26 plant morphological descriptors was confirmed. Surprisingly, the cardosin profiles of each genotype, based on four main groups A0, A1, A and B, were stable during the annual flower harvesting period and over all three years using ion-exchange chromatography and native-PAGE electrophoresis. This knowledge will allow an improvement in the quality and standardization of cardosin profiles from cardoon flowers used for cheese production and other innovative applications. The results obtained are promising for the development of a plant breeding program based on biochemical and morphological characteristics in order to obtain the most adapted plant architecture for combined purposes related to specific cardosins composition, flower and plant biomass production, and ease of harvesting.

Comparative Evaluation of the Antioxidant and Anti-Alzheimer's Disease Potential of Coumestrol and Puerarol Isolated from Pueraria lobata Using Molecular Modeling Studies.

The current study assesses the antioxidant effects of two similar isoflavonoids isolated from Pueraria lobata, coumestrol and puerarol, along with the cholinergic and amyloid-cascade pathways to mitigate Alzheimer's disease (AD). Antioxidant activity was evaluated via 1,1-diphenyl-2-picryhydrazyl (DPPH) and peroxynitrite (ONOO-) scavenging ability further screened via ONOO--mediated nitrotyrosine. Similarly, acetyl- and butyrylcholinesterase (AChE/BChE) and ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitory activities were assessed together with docking and kinetic studies. Considering DPPH and ONOO- scavenging activity, coumestrol (EC50 values of 53.98 and 1.17 µM) was found to be more potent than puerarol (EC50 values of 82.55 and 6.99 µM) followed by dose dependent inhibition of ONOO--mediated nitrotyrosine. Coumestrol showed pronounced AChE and BChE activity with IC50 values of 42.33 and 24.64 µM, respectively, acting as a dual cholinesterase (ChE) inhibitor. Despite having weak ChE inhibitory activity, puerarol showed potent BACE1 inhibition (28.17 µM). Kinetic studies of coumestrol showed AChE and BChE inhibition in a competitive and mixed fashion, whereas puerarol showed mixed inhibition for BACE1. In addition, docking simulations demonstrated high affinity and tight binding capacity towards the active site of the enzymes. In summary, we undertook a comparative study of two similar isoflavonoids differing only by a single aliphatic side chain and demonstrated that antioxidant agents coumestrol and puerarol are promising, potentially complementary therapeutics for AD.

BACE1 molecular docking and anti-Alzheimer's disease activities of ginsenosides.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginsenosides are natural product steroid glycosides and triterpene saponins obtained from the Panax species. Panax ginseng has been widely used as a traditional Chinese medicine (TCM) for around a thousand years, especially in East Asian countries. Ginseng, the root and rhizome of the most popular species P. ginseng, used as tonic, prophylactic agent and restorative. In TCM, ginseng is highly valued herb and has been applied to a variety of pathological conditions and illnesses such as hypodynamia, anorexia, shortness of breath, palpitation, insomnia, impotence, hemorrhage and diabetes. AIM OF THE STUDY: The basic aim of this study was to evaluate the anti-Alzheimer's disease activities of selected ginsenosides (Rb1, Rb2, Rc, Re, Rg1, and Rg3) according to peroxynitrite (ONOO(‒)) scavenging activity and inhibitory activity of ONOO(-)-mediated nitrotyrosine formation as a measure of changes in oxidative stress. In addition, molecular docking simulation studies were performed to predict binding energies of the ginsenosides with ß-site amyloid precursor protein cleaving enzyme 1 (BACE1, ß-secretase) and identify the interacting residues. MATERIALS AND METHODS: In vitro cholinesterase enzyme assays by using acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and BACE1 were performed. In vitro authentic peroxynitrite scavenging activity and inhibitory activity against ONOO(-)-mediated nitrotyrosine formation were also performed. Molecular docking simulation studies were performed with Autodock Vina software and Discovery studio 4.1. RESULTS: In vitro enzyme assays demonstrated that ginsenosides have significant inhibitory potential against AChE, BChE, and BACE1, as well as ONOO(-) and nitrotyrosine formation. Most importantly, significant AChE inhibitory activities were observed for Re; BChE for Rg3; and BACE1 for Rc, with IC50 values of 29.86±3.20, 16.80±0.36, and 59.81±2.74µg/mL, respectively. Among the tested ginsenosides, Rb1 exhibited a higher scavenging activity against ONOO(-) with an IC50 value of 27.86±1.34µg/mL, while Rc and Rg3 exhibited impressive inhibitory activity against the formation of nitrotyrosine. In addition, molecular docking studies revealed potential BACE1 inhibitory activity of ginsenosides, especially Rb1 and Rb2, which exhibited good binding affinities towards BACE1, with docking scores of -10kcal/mol. CONCLUSION: The findings of the present study suggest the potential of ginsenosides (Rb1, Rb2, Rc, Re, Rg1, and Rg3) for use in the development of therapeutic or preventive agents for Alzheimer's disease, especially through inhibition of AChE, BChE and BACE1 activities, as well as scavenging of ONOO(-) and inhibition of nitrotyrosine formation.

Isolation of cholinesterase and ß-secretase 1 inhibiting compounds from Lycopodiella cernua.

Three new serratene-type triterpenoids (1-3) and a new hydroxy unsaturated fatty acid (13) together with nine known compounds (4-12) were isolated from Lycopodiella cernua. The chemical structures were established using NMR, MS, and Mosher's method. Compound 13 showed the most potent inhibitory activity against acetylcholinesterase (AChE) with an IC50 value of 0.22µM. For butyrylcholinesterase (BChE) inhibitory activity, 5 showed the most potent activity with an IC50 value of 0.42µM. Compound 2 showed the most potent activity with an IC50 of 0.23µM for BACE-1 inhibitory activity. The kinetic activities were investigated to determine the type of enzyme inhibition involved. The types of AChE inhibition shown by compounds 4, 5, and 13 were mixed; BChE inhibition by 5 was competitive, while 2 and 6 showed mixed-types. In addition, molecular docking studies were performed to investigate the interaction of these compounds with the pocket sites of AChE. The docking results revealed that the tested inhibitors 3, 4, and 13 were stably present in several pocket domains of the AChE residue.

Partial Molecular Characterization of Arctium minus Aspartylendopeptidase and Preparation of Bioactive Peptides by Whey Protein Hydrolysis.

In this article, we report the cloning of an aspartic protease (AP) from flowers of Arctium minus (Hill) Bernh. (Asteraceae) along with the use of depigmented aqueous flower extracts, as a source of APs, for the hydrolysis of whey proteins. The isolated cDNA encoded a protein product with 509 amino acids called arctiumisin, with the characteristic primary structure organization of typical plant APs. Bovine whey protein hydrolysates, obtained employing the enzyme extracts of A. minus flowers, displayed inhibitory angiotensin-converting enzyme (ACE) and antioxidant activities. Hydrolysates after 3 and 5 h of reaction (degree of hydrolysis 2.4 and 5.6, respectively) and the associated peptide fraction with molecular weight below 3 kDa were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization/time of flight mass spectrometry, and reverse phase-high-performance liquid chromatography. The results obtained in this study demonstrate the viability of using proteases from A. minus to increase the antioxidant and inhibitory ACE capacity of whey proteins.

In Silico Design of Beta-Secretase Inhibitors in Alzheimer's Disease.

Alzheimer's disease is the major cause of senile dementia, flewing out 10% of 65 years old and 50% of 85 years old global population. The major fisiopathologic characteristics of Alzheimer's disease are the deposition of extracellular neuritic plaques and the presence of intracellular neurofibrillary tangles in memory-related areas of the brain. The plaques are composed by the ß-amyloid peptide with 40 or 42 residues, result from hydrolysis of the amyloid precursor protein by the ß-secretase 1 (BACE-1) on the amyloidogenic pathway, that begins with the BACE-1 and which inhibition is considered one of the most promising treatments available of Alzheimer's disease. In this work, molecular modeling techniques such as virtual screening of compound libraries, pharmacophore-based virtual screening, molecular interaction fields, ADME/Tox predictions, and similarity search were used to design novel inhibitors of BACE-1, starting from structures available in the Protein Data Bank. The results obtained from all virtual screening techniques leaded to 10 promising compounds, which were then evaluated by enzymatic assays, and, three of them showed inhibitory activity of BACE-1 at a 1 µm range.

Computer-aided structure-based design of multitarget leads for Alzheimer's disease.

Alzheimer's disease is a neurodegenerative pathology with unmet clinical needs. A highly desirable approach to this syndrome would be to find a single lead that could bind to some or all of the selected biomolecules that participate in the amyloid cascade, the most accepted route for Alzheimer disease genesis. In order to circumvent the challenge posed by the sizable differences in the binding sites of the molecular targets, we propose a computer-assisted protocol based on a pharmacophore and a set of required interactions with the targets that allows for the automated screening of candidates. We used a combination of docking and molecular dynamics protocols in order to discard nonbinders, optimize the best candidates, and provide a rationale for their potential as inhibitors. To provide a proof of concept, we proceeded to screen the literature and databases, a task that allowed us to identify a set of carbazole-containing compounds that initially showed affinity only for the cholinergic targets in our experimental assays. Two cycles of design based on our protocol led to a new set of analogues that were synthesized and assayed. The assay results revealed that the designed inhibitors had improved affinities for BACE-1 by more than 3 orders of magnitude and also displayed amyloid aggregation inhibition and affinity for AChE and BuChE, a result that led us to a group of multitarget amyloid cascade inhibitors that also could have a positive effect at the cholinergic level.

Oleic acid and linoleic acid from Tenebrio molitor larvae inhibit BACE1 activity in vitro: molecular docking studies.

In our ongoing research to find therapeutic compounds for Alzheimer's disease (AD) from natural resources, the inhibitory activity of the BACE1 enzyme by Tenebrio molitor larvae and its major compounds were evaluated. The T. molitor larvae extract and its fractions exhibited strong BACE1 suppression. The major components of hexane fraction possessing both high yield and strong BACE1 inhibition were determined by thin layer chromatography, gas chromatography, and nuclear magnetic resonance analysis. A remarkable composition of unsaturated long chain fatty acids, including oleic acid and linoleic acid, were identified. Oleic acid, in particular, noncompetitively attenuated BACE1 activity with a half-maximal inhibitory concentration (IC50) value of 61.31 µM and Ki value of 34.3 µM. Furthermore, the fatty acids were stably interacted with BACE1 at different allosteric sites of the enzyme bound with the OH of CYS319 and the NH3 of TYR320 for oleic acid and with the C=O group of GLN304 for linoleic acid. Here, we first revealed novel pharmacophore features of oleic acids and linoleic acid to BACE1 by in silico docking studies. The present findings would clearly suggest potential guidelines for designing novel BACE1 selective inhibitors.

Identification of BACE1 inhibitors from Panax ginseng saponins-An Insilco approach.

BACE1, a ß secretase candidate enzyme, initiates the Alzheimer's disease (AD) pathogenesis via amyloid ß (Aß) peptide production serving as a potential therapeutic target. Previous experimental evidence suggested that ginsenosides, a key component of Panax ginseng, are effective against AD. In this study, we implemented a molecular modeling method to reveal the inhibitory action of ginsenosides on BACE1 activity. We selected 12 ginsenosides and performed molecular docking studies to evaluate its interaction with the BACE1 active site, which is essential for inhibition. Further ADMET filtration was applied to find drug-like molecules with a specific ability to cross blood brain barrier (BBB), and to determine toxicity. The BACE1-ginsenosides complex was further subjected to a molecular dynamics simulation to study the stability of the complex and its hydrogen bond interactions. In summary, our findings show ginsenosides CK, F1, Rh1 and Rh2 are potential BACE1 inhibitors from Panax ginseng.

Design, synthesis and evaluation of 3-(2-aminoheterocycle)-4-benzyloxyphenylbenzamide derivatives as BACE-1 inhibitors.

Three series of 3-(2-aminoheterocycle)-4-benzyloxyphenylbenzamide derivatives, 2-aminooxazoles, 2-aminothiazoles, and 2-amino-6H-1,3,4-thiadizines were designed, synthesized and evaluated as ß-secretase (BACE-1) inhibitors. Preliminary structure-activity relationships revealed that the existence of a 2-amino-6H-1,3,4-thiadizine moiety and α-naphthyl group were favorable for BACE-1 inhibition. Among the synthesized compounds, 5e exhibited the most potent BACE-1 inhibitory activity, with an IC50 value of 9.9 µΜ and it exhibited high brain uptake potential in Madin-Darby anine kidney cell lines (MDCK) and a Madin-Darby canine kidney-multidrug resistance 1 (MDCK-MDR1) model.

Modulation of in vitro activity of zymogenic and mature recombinant human ß-secretase by dietary plants.

The in vitro activity of human recombinant ß-secretase (BACE1) was studied using a fluorogenic substrate based on the cleavage site for the enzyme in the Swedish mutation of amyloid precursor protein. The enzyme was inhibited by a control peptide inhibitor with good repeatability. The enzyme preparation comprised a mixture of pro-enzyme or zymogen and mature enzyme whereby the pro-enzyme sequence forms a 'flap' that can obstruct the binding site. 'Open flap' forms of the zymogen and mature enzyme are active, but the 'closed flap' form of the zymogen is inactive. This mixture of enzyme populations permitted apparent stimulation of enzyme activity under particular conditions, presumably due to facilitating flap-opening of the zymogen. As reported for heparin, enzyme activation was stimulated in the presence of low concentrations of Tween 20 and dimethylsulfoxide before becoming inhibited at higher concentrations. Dietary plant extracts either consistently inhibited (e.g. clove, tea, cinammon) or consistently stimulated (e.g. mushroom, parsley, asparagus) BACE1. Common structural features identified by Fourier transform infrared spectroscopy revealed that BACE1 activity could be explained by differential interactions of either small molecule or polymeric species with mature versus zymogen forms of the enzyme, respectively. Further, enzyme activity could be reversed by mixtures of high and low mass species. These results may have implications for the regulation of ß-secretase activity in vivo by either endogenous or possibly dietary factors and for a potential role of BACE1 in stimulation of the production of amyloid beta peptide in sporadic Alzheimer's disease.

Characterisation of hydrazides and hydrazine derivatives as novel aspartic protease inhibitors.

Virtual screening of an in-house virtual library of synthetic compounds using FlexX, followed by enzyme inhibition, identified hydrazide and hydrazine derivatives as novel aspartic protease inhibitors. These compounds inhibited human cathepsin D and Plasmodium falciparum plasmepsin-II with low micromolar concentrations (IC(50) = 1-2.5 microM). Modelling studies with plasmepsin-II predicted binding of ligands at the centre of the extended substrate-binding cleft, where hydrazide/hydrazine parts of the inhibitors acted as the transition state mimic by forming electrostatic interactions with catalytic aspartates.