3D-pharmacophore model based virtual screening to identify dual-binding site and selective acetylcholinesterase inhibitors

The formation of β-amyloid plaques in the brain is a key neurodegenerative event in Alzheimer’s disease (AD). Interestingly, research on acetylcholinesterase (AChE) enzyme has increased due to findings supporting this enzyme involvement in the β-amyloid peptide fibril formation during AD pathogenesis. In this investigation, chemical features based 3D pharmacophore models were developed from structurally diverse xanthostigmine derivatives, known inhibitors of AChE enzyme, using 3D-QSAR pharmacophore generation module in Discovery Studio2.5 (DS2.5). The constructed pharmacophore models for AChE inhibitors was further cross-validated using test set and Cat-Scramble methodology. The best quantitative pharmacophore model Hypo1, was used for screening the chemical databases of small compounds including Specs, NCI, and IBScreen, to identify the new compounds that are presumably able to act as dual-binding site AChE inhibitors. The screened virtual hits were then subjected to the Lipinski’s rule of five, blood–brain barrier (BBB), PSA, LogS, percent human oral absorption, and toxicity analysis. Finally, 32 compounds were identified as potential leads against AChE enzyme, showing good estimated activities and promising ADMET properties. Molecular docking of these compounds using FlexX software showed catalytic and peripheral anionic binding site interactions, so called dual binding of the AChE enzyme. Docking study was also performed on butyrylcholinesterase in order to understand the compound selectivity. This study may assist in the discovery and design of novel dual binding site and selective AChE inhibitors with potent inhibitory activity.     

A further approach to quantitative determinations by thin layer chromatography.

In the proposed method, an internal standard, suitably chosen, is added in equal quantities to the sample was well as to the standard solutions. A standard curve is obtained by plotting the ratio Ds/Di for each of the standard solutions against the logarithm of the quantity of material present in each solution, where Ds and Di are the average diameters of the standard and internal standard spots, respectively. It was disclosed that the ratio Ds/Di for a given solution is constant, regardless of the volume of the solution spotted. As a result, no exact equal known volumes are needed for spotting. The method has been tested with estimation of menthol in volatile oil of peppermint and strychine in nux vomica seed.     

Advanced screening assays to rapidly identify solubility-enhancing formulations: high-throughput, miniaturization and automation

Development of solubility-enhancing formulations for poorly water-soluble compounds always poses a challenge. Conventional formulation screening assays are potentially time-consuming and labor-intensive and, moreover, require a large amount of a compound; they are not ideal when compound availability and testing time are limited. In recent years, in-vitro screening assays that are rapid, inexpensive, minimally labor-intensive, and require only small quantities of a compound have become available. These advanced assays allow high-throughput automation, miniaturization, and parallel processing, thereby enabling scientists to rapidly identify solubility-enhancing formulations with milligram or sub-milligram quantities of an active pharmaceutical ingredient (API). This article reviews these assays for rapidly screening the aqueous solubility of lead compounds and the solubility-enhancing formulations with limited quantities of API.     

Virtual screening to successfully identify novel janus kinase 3 inhibitors: a sequential focused screening approach

In an effort to identify novel Janus kinase 3 inhibitors, a sequential focused screening approach was adopted to search our in-house chemical database. By biologically testing only 79 selected compounds, we successfully identified 19 compounds showing IC 50 < 20 microM, with four of them in the nanomolar range. Particularly, a 3,5-disubstituted pyrazolo[4,3- d]pyrimidine scaffold emerged as a promising candidate for further lead optimization. With the advantages of efficiency and flexibility, this approach may be utilized to identify leads for other therapeutic targets.     

薄层层离法在研究天然产物中的应用 Ⅴ.香豆酮类化合物的鉴定及分离

本文选择了6种香豆酮化合物(其中5种为呋喃香豆素),对不含粘合剂氧化鋁薄层层离法的一些条件进行了研究。結果采用細度小于160号篩孔,活性2—3級的酸性氧化鋁为吸附剂;石油醚:氯仿(1:1)、石油醚:二氧六环(10:2)、石油醚:乙醇(1:1)等溶剂为推进剂。在推进前不必进行蒸气飽和。此法可以应用于一些香豆酮化合物的鉴定和小量分离,并应用于前胡和藁本研究工作而获得一定結果。     

In vitro flow cytometry method to quantitatively assess inhibitors of P-glycoprotein.

P-glycoprotein (Pgp)-mediated drug efflux is a major factor contributing to the variance of absorption and distribution of many drugs. A simple and reliable in vitro method to identify inhibitors of Pgp helps to prevent the potential of drug interactions. Using daunorubicin as a fluorescent marker and vanadate as a positive control compound, a functional flow cytometry method for assessing the ability of a drug to inhibit Pgp-mediated drug efflux from CR1R12 multidrug-resistant cells has been evaluated. Quantitation of the relative fluorescence was used to compare potency of individual inhibitors. Known Pgp inhibitors, such as cyclosporin A, nicardipine, verapamil, quinidine, terfenadine, tamoxifen, and vinblastine were demonstrated to inhibit the Pgp-mediated efflux of daunorubicin. Cyclosporin A and terfenadine were the most potent inhibitors among the compounds tested. Tetraphenylphosphonium and alpha-tocopherol had little inhibitory effect. Progesterone produced significant inhibition at relatively high concentrations. This study demonstrated that this in vitro flow cytometry method is a simple, sensitive, and quantitative tool to assess the capacity of a drug to inhibit Pgp transporters, and is useful for screening or identifying inhibitors of Pgp as well as evaluation of potential for drug interactions.     

In vitro binding of oxime acetylcholinesterase reactivators to proteoglycans synthesized by cultured chondrocytes and fibroblasts.

The incorporation of the 14C-labelled acetylcholinesterase reactivators 1-(methyl-imidazolium)-3 (4-carbaldoxime-pyridinium) propane dibromide (pyrimidoxime) and N,N'-trimethylene bis(pyridinium-4-aldoxime) dibromide (TMB4) into cultured chondrocytes and fibroblasts was measured and their binding to macromolecules synthesized by these cells studied. The results showed that these drugs concentrated slowly and poorly into these cells, but bound firmly to high molecular mass materials in the culture supernatants. The chromatographic properties of these macromolecules on Sepharose CL-2B in non-dissociative or dissociative conditions were similar to those of the proteoglycans synthesized by these cells. Dialysis of the macromolecule-bound drugs against increasing pH buffers showed half-dissociation pH > 8, identical to those for chondroitin sulphate. These results suggest strongly that pyrimidoxime and TMB4 are bound to proteoglycans by ionic interactions, and this together with their poor lipophilicity can explain their high selectivity for the cartilaginous tissues as opposed to other proteoglycan-containing structures such as skin.     

Facile in vitro method for screening inhibitors of IgE binding to mast cells.

A method for rapidly testing large numbers of chemical structures as potential modulators of the interaction between immunoglobulin E (IgE) and its specific receptors on rat peritoneal mast cells is described. IgE, isolated from the ascitic fluid of a transplantable rat IgE immunocytoma, is labeled with iodine-125 under mild conditions employing the Bolton--Hunter reagent. The antibody is incubated with mixed periotoneal cells at 37 degrees, and the cell-bound IgE is separated from unbound label by sedimentation through an 8% sucrose--polymer solution in microsediment tubes. Optimal conditions for the interaction of 3 nM IgE with 3 X 10(5) mast cells in 150 mul are: incubation time, 2 hr; pH, 6.5--7.0; and ionic strength, equivalent to 150 mM NaCl. Mixed peritoneal cells bind IgE with an affinity equal to that of purified mast cells. Human IgE pentapeptide III and several antiallergic agents do not compete with rat IgE in this assay.     

A rapid thin layer chromatographic procedure to identify poor and extensive oxidative drug metabolizers in man using dextromethorphan.

A rapid TLC method is presented to distinguish poor oxidative drug metabolizers from extensive oxidative drug metabolizers. Dextromethorphan (1) is used as test probe because it is safe, well characterized, generally available and easy to measure. The method is based on the extraction of 1 and its major oxidative metabolite dextrorphan (2) from urine, followed by separation on a TLC plate and visualized by a combined Marquis/Mandelin reaction. The intensities of the spots are then compared with a series of standard mixtures containing 1 and 2 in a ratio of 0.3 at different concentrations. This value represents the antimode that separates poor from extensive metabolizers, and an individual is identified as a poor metabolizer if the intensity ratio of the two spots from his urine sample is higher than 0.3. The proposed TLC method was cross checked with an HPLC method and found to correctly identify 9 poor metabolizers out of a population of 71 volunteers.     

In vitro methods to study chemically-induced hepatotoxicity: a literature review

Understanding the hepatotoxicity of drugs and chemicals is essential for progress in the pharmaceutical industry, medical science and academic research. The study of hepatotoxicity in vitro is complicated by the difficulty of maintaining hepatocytes in culture due to a lack of understanding of the humoral and matrix requirements of these cells. A variety of in vitro models of the liver have been developed, such as perfused livers, liver slices and three-dimensional perfused bioreactors, but the static cell culture is the most commonly used system. In this review we present the advantages and disadvantages of each system and their roles in the study of hepatotoxicity. We will also discuss how the various culture conditions such as medium and matrix composition affect the systems. The technological advances, which started the fields of genomics, proteomics and metabonomics are playing a very important role in uncovering novel biochemical pathways and markers of toxicity. Several of these studies have focused on hepatotoxicity, particularly on the effects of acetaminophen, carbon tetrachloride and aflatoxin B1. Finally, we will discuss the new field of systems biology, which focuses on interpreting and integrating data from all of the other fields.     

Drugs-biomolecule interactions: binding study of substrate and inhibitors to acetylcholinesterase using NMR.

NMR was used to study the binding of acetylcholine, atropine, and physostigmine to acetylcholinesterase. Changes in the linewidth of the N-methyl resonance of acetylcholine, resulting from association with the enzyme during hydrolysis, were utilized to study the enzyme-substrate interaction. Physostigmine inhibited the binding of the substrate while atropine accelerated substrate hydrolysis without interfering with its binding. The dissociation constant, KD and the linewidth of the acetylcholinesterase-inhibitor complex, increment v bound, for atropine and physostigmine can be estimated from the linewidth changes of the N-methyl and phenyl group resonances of atropine and from the N-methyl and C-methyl group resonances of physostigmine resulting from association with the enzyme. The results indicate that there is at least one binding site on the enzyme surface for atropine and one for physostigmine. Further evidence that the two sites are distinct is indicated by the fact that gallamine displaces atropine from its site without competing with physostigmine.     

Aryl acylamidase of monkey brain and liver: response to inhibitors and relationship to acetylcholinesterase.

The serotonin sensitive aryl acylamidase (aryl acylamide amidohydrolase EC 3.5.1.13) of monkey brain was compared with the liver enzyme. Although the two enzymes showed some similarities in their properties such as pH optima, the effect of metal ions and thiol agents, they significantly differed in their mol. wt and response to inhibitors. The liver enzyme had a higher mol. wt as observed by gel filtration on Sepharose 6B and a greater heat stability. The brain enzyme was inhibited specifically by the amines serotonin and tryptamine as well as by acetylcholine and its analogues and homologues in a non-competitive manner. The liver enzyme was unaffected by the above mentioned amines or acetylcholine but it was non-competitively inhibited by indole-3-acetic acid and indole-3-propionic acid both compounds having no effect on the brain enzyme. Eserine, a strong competitive inhibitor of acetylcholinesterase, at 10^?7M inhibited the brain aryl acylamidase to 75 per cent leaving the liver enzyme unaffected. Eserine inhibition of the brain enzyme was non-competitive. From Dixon plots serotonin, acetylcholine and eserine were shown to act at the same site on brain aryl acylamidase. The inhibition of the brain enzyme by eserine and acetylcholine, the elution of both aryl acylamidase and acetylcholinesterase activities in the same fractions during gel filtration and the regional distribution of aryl acylamidase in the brain suggested the association of aryl acylamidase with acetylcholinesterase in the brain though not in the liver.     

Validation and application of Caco-2 assays for the in vitro evaluation of development candidate drugs as substrates or inhibitors of P-glycoprotein to support regulatory submissions

1.?An understanding of the role that transporters, in particular P-glycoprotein (P-gp), can play in the absorption, distribution, metabolism and excretion (ADME) of candidate drugs, and an assessment of how these processes might impact on toxicity and the potential for drug–drug interactions in the clinic, is required to support drug development and registration. It is therefore necessary to validate preclinical assays for the in vitro evaluation of candidate drugs as substrates or inhibitors of human P-gp.

2.?The present study has characterized a Caco-2 cell monolayer model by determining the bi-directional apparent permeabilities and efflux ratios of the known P-gp substrates ([³H]-digoxin, [³H]-ketoconazole, [³H]-verapamil, [³H]-quinidine, dipyridamole and loratidine; 1–100 µM) a non-substrate ([³H]-propranolol; 10 µM), or by determining the inhibitory potencies (IC₅₀) of inhibitors (verapamil, ketoconazole, quinidine, dipyridamole and probenecid; 0.1–100 µM) on the basolateral-to-apical transport of [³H]-digoxin (5 µM), in order to validate methodologies for the identification of substrates or inhibitors of P-gp, respectively.

3.?The reproducibility of the [³H]-digoxin or verapamil data determined from replicate monolayers across different cell passages indicates that the functional expression of P-gp is consistent across the range of passages (25–40) utilized for transport experiments and that the determination of bi-directional apparent permeability, or IC₅₀ for inhibition of P-gp, respectively, need only be performed on one occasion for a test compound. [³H]-digoxin and [³H]-propranolol or verapamil and probenecid were considered to be appropriate positive and negative controls of P-gp-mediated transport, or inhibition of P-gp, respectively, to ensure performance of the assays when assessing candidate drugs. Additionally, the low IC₅₀ values determined for ketoconazole and quinidine indicated that these inhibitors were suitable to use to confirm the role of P-gp in the efflux of a test compound.

4.?These validated Caco-2 assays are robust, reproducible and suitable for routine in vitro evaluation of candidate drugs. They have been successfully applied to development projects resulting in the identification of two candidate drugs as substrates and inhibitors of P-gp, whereas a third was neither a substrate nor an inhibitor of this transporter.