This document summarizes a study that evaluated the ability of functionalized polyamidoamine (PAMAM) dendrimers to capture organophosphate pesticides and chemical warfare agents. The researchers synthesized PAMAM G4 and G5 derivatives functionalized with biomolecules like folic acid, coumarin, arginine, lysine, and asparagine. They then used these compounds to adsorb the organophosphates azinphos-methyl and methamidophos. Both experimental assays using high-performance liquid chromatography and in silico molecular dynamics simulations supported the findings that PAMAM-G4-Asn had the highest binding capacity for methamidophos.
Este es uno de los estudios científicos, recientemente publicados, que se ha enfocado en registrar la cantidad del compuesto 4-Metilidimazol que se encuentra en las bebidas de Cola y en la cerveza oscura. El compuesto 4-Metilidimazol es un subproducto cancerígeno que se encuentra en el colorante Caramelo IV utilizado en estas bebidas. El estudio reconoce que la mayor ingesta que se da de este compuesto es a través de las bebidas de Cola. Estima que en los Estados Unidos el consumo promedio diario de bebidas de Cola puede representar una ingesta de 342 microgramos diarios de 4-Metilidimazol. Esta cantidad significa varias veces el nivel máximo recomendado como seguro de ingesta de este compuesto sugerido por la autoridad de California, para todo un día que es de 16 microgramos al día. El estudio reporta que una sola lata de bebida de Cola puede contener entre 12.95 y 214.55 microgramos de 4-Metilidimazol.
Hepatoprotective effect of ginger extract against the toxicity of 7, 12-dimet...Jing Zang
The present work was conducted to study the protective effect of ginger extract (GE) against the hepatotoxicity induced by 7, 12-dimethylbenz(a)anthracene (DMBA) in female rats. DMBA treated group showed a highly significant decrease in body weight. However, DMBA+GE treated group displayed a highly significant increase in body weight compared with DMBA treated group. Histologically, the liver of DMBA treated group showed nodule-like structures, hepatic cirrhosis, congestion of blood vessels, intercellular hemorrhage, hepatic pyknotic nuclei, lymphocytic infiltration, dilation of blood sinusoids and large amount of collagen fibres. DMBA+GE treated group displayed an improvement in the hepatic lesions induced by DMBA. Histochemically, the liver sections of DMBA treated group showed a marked depletion of polysaccharides and total protein content. However, DMBA+GE treated group showed a moderate increase in liver polysaccharides and total protein contents. Immunohistochemically, the hepatocytes of DMBA treated group showed a highly significant increase in the PCNA labelling index. However, DMBA+GE treated group displayed a highly significant decrease in the PCNA labelling index when compared with DMBA treated group.
Metabolomics is often described as the study of “the complete set of low molecular weight intermediates, which are context dependent, varying according to the physiology, developmental or pathological state of the cell, tissue, organ or organism”. In fact, metabolomics is a new term for an old science in which classical biochemical concepts are investigated. New and unique to the current research that is being conducted is the combination with genomics information and full system biology. In this refocus we will discuss the challenges in today's metabolomics research and how to address them
Este es uno de los estudios científicos, recientemente publicados, que se ha enfocado en registrar la cantidad del compuesto 4-Metilidimazol que se encuentra en las bebidas de Cola y en la cerveza oscura. El compuesto 4-Metilidimazol es un subproducto cancerígeno que se encuentra en el colorante Caramelo IV utilizado en estas bebidas. El estudio reconoce que la mayor ingesta que se da de este compuesto es a través de las bebidas de Cola. Estima que en los Estados Unidos el consumo promedio diario de bebidas de Cola puede representar una ingesta de 342 microgramos diarios de 4-Metilidimazol. Esta cantidad significa varias veces el nivel máximo recomendado como seguro de ingesta de este compuesto sugerido por la autoridad de California, para todo un día que es de 16 microgramos al día. El estudio reporta que una sola lata de bebida de Cola puede contener entre 12.95 y 214.55 microgramos de 4-Metilidimazol.
Hepatoprotective effect of ginger extract against the toxicity of 7, 12-dimet...Jing Zang
The present work was conducted to study the protective effect of ginger extract (GE) against the hepatotoxicity induced by 7, 12-dimethylbenz(a)anthracene (DMBA) in female rats. DMBA treated group showed a highly significant decrease in body weight. However, DMBA+GE treated group displayed a highly significant increase in body weight compared with DMBA treated group. Histologically, the liver of DMBA treated group showed nodule-like structures, hepatic cirrhosis, congestion of blood vessels, intercellular hemorrhage, hepatic pyknotic nuclei, lymphocytic infiltration, dilation of blood sinusoids and large amount of collagen fibres. DMBA+GE treated group displayed an improvement in the hepatic lesions induced by DMBA. Histochemically, the liver sections of DMBA treated group showed a marked depletion of polysaccharides and total protein content. However, DMBA+GE treated group showed a moderate increase in liver polysaccharides and total protein contents. Immunohistochemically, the hepatocytes of DMBA treated group showed a highly significant increase in the PCNA labelling index. However, DMBA+GE treated group displayed a highly significant decrease in the PCNA labelling index when compared with DMBA treated group.
Metabolomics is often described as the study of “the complete set of low molecular weight intermediates, which are context dependent, varying according to the physiology, developmental or pathological state of the cell, tissue, organ or organism”. In fact, metabolomics is a new term for an old science in which classical biochemical concepts are investigated. New and unique to the current research that is being conducted is the combination with genomics information and full system biology. In this refocus we will discuss the challenges in today's metabolomics research and how to address them
Simplified receptor based pharmacophore approach to retrieve potent ptp lar i...rajmaha9
Simplified Receptor Based Pharmacophore Approach to Retrieve Potent PTP-LAR Inhibitors Using Apoenzyme
M. Elizabeth Sobhia*
Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S.
Nagar, Punjab 160062, India
Abstract: The design of biological active compounds from the apoenzyme is still a challenging task. Herein a simple yet efficient technique is reported to generate a receptor based pharmacophore solely using a ligand-free protein crystal structure. Human leukocyte antigen-related phosphatase (PTP-LAR) is an apoenzyme and a receptor like transmembrane phosphatase that has emerged as a drug target for diabetes, obesity and cancer. The prior knowledge of the active residues responsible for the mechanism of action of the protein was used to generate the LUDI interaction map. Then, the complement negative image of the binding site was used to generate the pharmacophore features. A unique strategy was
followed to design a pharmacophore query maintaining crucial interactions with all the active residues, essential for the enzyme inhibition. The same query was used to screen several databases consisting of the Specs, IBS, iniMaybridge, NCI and an in-house PTP inhibitor databases. In order to overcome the common bioavailability problem associated with phosphatases, the hits obtained were filtered by Lipinski’s Rule of Five, SADMET properties and validated by docking studies in Glide and GOLD. These docking studies not only suggest the essential ligand binding interactions but also the binding patterns necessary for the LAR inhibition. The ligand pharmacophore mapping studies further validated the
screened protocol and supported that the final screened molecules, presumably, showed potent inhibitory activity.
Subsequently, these molecules were subjected to Derek toxicity predictions and nine new molecules with different
scaffold were obtained as non-toxic PTP-LAR inhibitors. The present prospective strategy is a powerful technique to
identify potent inhibitors using the protein 3D structure alone and is a valid alternative to other structure-based and
random docking approaches.
Expression, purification and spectroscopic characterization of the cytochrome...John Clarkson
K.J. McLean, M.R. Cheesman, S.L. Rivers, A. Richmond, D. Leys, S.K. Chapman, G.A. Reid, N.C. Price, S.M. Kelly, J. Clarkson, W.E Smith & A.W. Munro, “Expression, Purification and Spectroscopic Characterization of the Cytochrome P450 CYP121 from Mycobacterium Tuberculosis”, J. Inorganic Biochemistry, 91, 527-541, 2002.
Separation of L-Phenylalanine by Solvent Sublation and Solvent Extraction MethodBRNSS Publication Hub
Aims and Objectives: Separation and purification is a series of processes intended to isolate a single type of biomolecule from a complex mixture. Innovations in improvement of biodownstream processing, which is responsible for the separation of about 50–80% of recombinant proteins and other biomolecules, play a very important role in increasing the yield and reducing the cost of biopharmaceutical production. Methods: Biomolecule isolation and purification from a fermentation broth usually involve centrifugation, filtration, adsorption, and chromatography steps. Results and Discussions: Each step contributes to the product cost and product loss. Thus, we consider that solvent extraction and solvent sublation are the more economic processes for the separation of biomolecules. Conclusion: In extraction of phenylalanine, maximum extraction was observed at amino acid: surfactant ratio 1:1, amino acid: extractant ratio 1:1500, and pH at 3.1. The highest value of % recovery percentage and Co/Cw was 76.3 and 10.21, respectively. The main motive of this article is to provide the advantages of study on the solvent sublation and solvent extraction of l-phenylalanine over the other techniques.
Simplified receptor based pharmacophore approach to retrieve potent ptp lar i...rajmaha9
Simplified Receptor Based Pharmacophore Approach to Retrieve Potent PTP-LAR Inhibitors Using Apoenzyme
M. Elizabeth Sobhia*
Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S.
Nagar, Punjab 160062, India
Abstract: The design of biological active compounds from the apoenzyme is still a challenging task. Herein a simple yet efficient technique is reported to generate a receptor based pharmacophore solely using a ligand-free protein crystal structure. Human leukocyte antigen-related phosphatase (PTP-LAR) is an apoenzyme and a receptor like transmembrane phosphatase that has emerged as a drug target for diabetes, obesity and cancer. The prior knowledge of the active residues responsible for the mechanism of action of the protein was used to generate the LUDI interaction map. Then, the complement negative image of the binding site was used to generate the pharmacophore features. A unique strategy was
followed to design a pharmacophore query maintaining crucial interactions with all the active residues, essential for the enzyme inhibition. The same query was used to screen several databases consisting of the Specs, IBS, iniMaybridge, NCI and an in-house PTP inhibitor databases. In order to overcome the common bioavailability problem associated with phosphatases, the hits obtained were filtered by Lipinski’s Rule of Five, SADMET properties and validated by docking studies in Glide and GOLD. These docking studies not only suggest the essential ligand binding interactions but also the binding patterns necessary for the LAR inhibition. The ligand pharmacophore mapping studies further validated the
screened protocol and supported that the final screened molecules, presumably, showed potent inhibitory activity.
Subsequently, these molecules were subjected to Derek toxicity predictions and nine new molecules with different
scaffold were obtained as non-toxic PTP-LAR inhibitors. The present prospective strategy is a powerful technique to
identify potent inhibitors using the protein 3D structure alone and is a valid alternative to other structure-based and
random docking approaches.
Expression, purification and spectroscopic characterization of the cytochrome...John Clarkson
K.J. McLean, M.R. Cheesman, S.L. Rivers, A. Richmond, D. Leys, S.K. Chapman, G.A. Reid, N.C. Price, S.M. Kelly, J. Clarkson, W.E Smith & A.W. Munro, “Expression, Purification and Spectroscopic Characterization of the Cytochrome P450 CYP121 from Mycobacterium Tuberculosis”, J. Inorganic Biochemistry, 91, 527-541, 2002.
Separation of L-Phenylalanine by Solvent Sublation and Solvent Extraction MethodBRNSS Publication Hub
Aims and Objectives: Separation and purification is a series of processes intended to isolate a single type of biomolecule from a complex mixture. Innovations in improvement of biodownstream processing, which is responsible for the separation of about 50–80% of recombinant proteins and other biomolecules, play a very important role in increasing the yield and reducing the cost of biopharmaceutical production. Methods: Biomolecule isolation and purification from a fermentation broth usually involve centrifugation, filtration, adsorption, and chromatography steps. Results and Discussions: Each step contributes to the product cost and product loss. Thus, we consider that solvent extraction and solvent sublation are the more economic processes for the separation of biomolecules. Conclusion: In extraction of phenylalanine, maximum extraction was observed at amino acid: surfactant ratio 1:1, amino acid: extractant ratio 1:1500, and pH at 3.1. The highest value of % recovery percentage and Co/Cw was 76.3 and 10.21, respectively. The main motive of this article is to provide the advantages of study on the solvent sublation and solvent extraction of l-phenylalanine over the other techniques.
Georgian Women 2011: Early analysis from the 2011 Caucasus Barometer by Katy Pearce is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.
Based on a work at www.katypearce.net.
Permissions beyond the scope of this license may be available at http://www.katypearce.net/cv/georgia.
The use of agrochemicals has increased considerably in recent years, and consequently, there has been increased exposure of ecosystems and human populations to these highly toxic compounds. The study and development of methodologies to detect these substances with greater sensitivity has become extremely relevant. This article describes, for the first time, the use of atomic force spectroscopy (AFS) in the detection of enzyme-inhibiting herbicides. A nanobiosensor based on an atomic force microscopy (AFM) tip functionalised with the acetolactate synthase (ALS) enzyme was developed and characterised. The herbicide metsulfuron-methyl, an ALS inhibitor, was successfully detected through the acquisition of force curves using this biosensor. The adhesion force values were considerably higher when the biosensor was used. An increase of ~250% was achieved relative to the adhesion force using an unfunctionalised AFM tip. This considerable increase was the result of a specific interaction between the enzyme and the herbicide, which was primarily responsible for the efficiency of the nanobiosensor. These results indicate that this methodology is promising for the detection of herbicides, pesticides, and other environmental contaminants.
Murdannia loriformis(hassk) - Yapakking Suphalada C.
Beijing herbs grass to support such research out of the grass, the herb Beijing to treat cancer. No significant toxicity. When doing research, it was found that the in vitro anti-cancer The colon cancer and breast cancer cells. Were found to contain glycoprotein Goliath Sphinx Rapids (G 1 b) moderately inhibited the cancer cell. Beijing has also been found that grass. Stimulate immunity. Make you strong. Found that patients treated for eating grass, Beijing on the current pattern. Patients suffering from the side effects of lower radiation and Kate's Modern Therapy. Inhibit the spread of cancer and came back again. Including the adaptive immune system of the body.
The herbicide residue from intensive agricultural
activity provokes environmental disturbances and human health injuries. Among the enzymatic disruptor herbicides, mesotrione is able to inhibit 4-hydroxyphenylpyruvate dioxygenase (HPPD), which plays a key role in the carotenoid synthesis. Therefore, enzyme-based sensors are innovative options for monitoring herbicides used in agriculture.
The Karolinska Institute (KI) is the largest centre for medical education and research in Sweden and the home of the Nobel Prize in Physiology or Medicine.
KI consists of 22 departments and 600 research groups dedicated to improving human health through research and higher education.
The role of the Kohonen/Grafström team has been to guide the application, analysis, interpretation and storage of so called “omics” technology-derived data within the service-oriented subproject “ToxBank”.
Cloning and Expression of Outer Membrane Protein Omp38 Derived from Aeromonas hydrophila in Escherichia coli
http://dx.doi.org/10.21276/SSR-IIJLS.2019.5.3.8
Novel 1,3,4-Thiadiazole Linked Amide Derivatives of Pteridone: Synthesis and ...Ratnakaram Venkata Nadh
Cancer is a second leading cause of death after heart attack, in developing as well as undeveloped
countries. It is caused by unregulated growth and metastasis of the abnormal cancer cells.
Cancer can be cured by radiation, immunotherapy and chemotherapy, among them; chemotherapy is a
good treatment for cancer, in which chemotherapeutic drug is used. The anticancer activity of newly
synthesized compounds (13a-j) was carried out on four different types of human cancer cell lines like
MCF-7 (breast), A549 (lung), Colo-205 (colon) and A2780 (ovarian) by the MTT method, and compared
to etoposide used as a positive control. Among them, compound 13g with electron-withdrawing
(3,5-dinitro) group, exhibited more promising activity in all cell lines (MCF-7 = 0.10±0.076 μM, A549
= 0.17±0.039 μM, Colo-205= 0.13±0.022 μM and A2780 = 0.87±0.027μM). This compound may act
as lead drug in cancer chemotherapy. In future, this compound can be examined for clinical studies.
A STUDY TO EVALUATE THE IN VITRO ANTIMICROBIAL ACTIVITY AND ANTIANDROGENIC E...Dr. Pradeep mitharwal
The present paper deals with synthesis and characterization
of some new chromium (III) Schiff base complexes using microwave irradiation
technique as well as conventional heating. The S∩N donor benzothiazolines, 1-
(2-furanyl) ethanone benzothiazoline (Bzt1N
∩
SH), 1-(2-thienyl) ethanone
benzothiazoline (Bzt2N
∩
SH) and 1-(2-pyridyl) ethanone benzothiazoline
(Bzt3N
∩
SH) were prepared by the condensation of ortho-aminothiophenol with
respective ketones in ethanol.
ABSTRACT- The anticancer drug arsenic trioxide is effective for acute promyelocytic leukemia. But the clinical trials are
restricted due to its potential side effects. Since the major part of arsenic metabolism and detoxification occurs in liver,
this organ faces the major threat. The hepatic side effects include fatty liver, fibrosis, and inflammation and hepatocyte
degeneration. Our study aimed to evaluate the protective potential of the fatty acid, docosahexaenoic acid, against adversities
of arsenic trioxide in an in vitro model, the Chang liver cells. Two preliminary dose standardization assays, cell
viability and lactate dehydrogenase release assays, were employed. The assays were performed as Pre-treatment,
Co-treatment and Post treatment experiments for a period of 24 hours. Arsenic trioxide at various doses (2.5, 5, 7.5, 10,
12.5 and 15 μM) showed a significant (p≤0.05) dose dependant reduction in cell viability along with a dose dependant
enhancement of lactate dehydrogenase release. However when the cells were treated with a combination of docosahexaenoic
acid at varying concentrations (50, 75, 100, 125 and 150 μM), the above mentioned conditions were found to be
reversed in Pre-treatment and Co-treatment experiments, but not in Post treatment. The most effective combination was
found to be 10 μM arsenic trioxide with 100 μM of docosahexaenoic acid in both Pre-treatment and Co- treatment studies.
Thus the preliminary assays of our study showed that docosahexaenoic acid administration as Pre-treatment or
Co-treatment can aid in reducing arsenic trioxide induced hepatotoxicity. Further studies are required to elucidate the mechanisms
behind the protective effects.
Key Words– Arsenic trioxide, hepatotoxicity, docosahexaenoic acid, cell damage
Salas, V. (2024) "John of St. Thomas (Poinsot) on the Science of Sacred Theol...Studia Poinsotiana
I Introduction
II Subalternation and Theology
III Theology and Dogmatic Declarations
IV The Mixed Principles of Theology
V Virtual Revelation: The Unity of Theology
VI Theology as a Natural Science
VII Theology’s Certitude
VIII Conclusion
Notes
Bibliography
All the contents are fully attributable to the author, Doctor Victor Salas. Should you wish to get this text republished, get in touch with the author or the editorial committee of the Studia Poinsotiana. Insofar as possible, we will be happy to broker your contact.
ANAMOLOUS SECONDARY GROWTH IN DICOT ROOTS.pptxRASHMI M G
Abnormal or anomalous secondary growth in plants. It defines secondary growth as an increase in plant girth due to vascular cambium or cork cambium. Anomalous secondary growth does not follow the normal pattern of a single vascular cambium producing xylem internally and phloem externally.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
ESR spectroscopy in liquid food and beverages.pptxPRIYANKA PATEL
With increasing population, people need to rely on packaged food stuffs. Packaging of food materials requires the preservation of food. There are various methods for the treatment of food to preserve them and irradiation treatment of food is one of them. It is the most common and the most harmless method for the food preservation as it does not alter the necessary micronutrients of food materials. Although irradiated food doesn’t cause any harm to the human health but still the quality assessment of food is required to provide consumers with necessary information about the food. ESR spectroscopy is the most sophisticated way to investigate the quality of the food and the free radicals induced during the processing of the food. ESR spin trapping technique is useful for the detection of highly unstable radicals in the food. The antioxidant capability of liquid food and beverages in mainly performed by spin trapping technique.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
The use of Nauplii and metanauplii artemia in aquaculture (brine shrimp).pptxMAGOTI ERNEST
Although Artemia has been known to man for centuries, its use as a food for the culture of larval organisms apparently began only in the 1930s, when several investigators found that it made an excellent food for newly hatched fish larvae (Litvinenko et al., 2023). As aquaculture developed in the 1960s and ‘70s, the use of Artemia also became more widespread, due both to its convenience and to its nutritional value for larval organisms (Arenas-Pardo et al., 2024). The fact that Artemia dormant cysts can be stored for long periods in cans, and then used as an off-the-shelf food requiring only 24 h of incubation makes them the most convenient, least labor-intensive, live food available for aquaculture (Sorgeloos & Roubach, 2021). The nutritional value of Artemia, especially for marine organisms, is not constant, but varies both geographically and temporally. During the last decade, however, both the causes of Artemia nutritional variability and methods to improve poorquality Artemia have been identified (Loufi et al., 2024).
Brine shrimp (Artemia spp.) are used in marine aquaculture worldwide. Annually, more than 2,000 metric tons of dry cysts are used for cultivation of fish, crustacean, and shellfish larva. Brine shrimp are important to aquaculture because newly hatched brine shrimp nauplii (larvae) provide a food source for many fish fry (Mozanzadeh et al., 2021). Culture and harvesting of brine shrimp eggs represents another aspect of the aquaculture industry. Nauplii and metanauplii of Artemia, commonly known as brine shrimp, play a crucial role in aquaculture due to their nutritional value and suitability as live feed for many aquatic species, particularly in larval stages (Sorgeloos & Roubach, 2021).
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
2. Durán-Lara et al. 581Vol. 26, No. 3, 2015
selective capture of toxic compounds in vivo. The aim of
this study is to evaluate the ability of dendrimers to capture
OPs in a selective manner. Polyamidoamine (PAMAM)
dendrimers are a class of artificial macromolecules that
have shown excellent potential in biomedical applications
due to their unique physical and chemical properties.
Among these properties, PAMAM show a precise control
on molecular size, shape and density, high solubility, a
large number of surface functional groups and hydrophobic
cavities that can capture molecules of interest.13
PAMAM
are biocompatible, non-immunogenic, water-soluble, and
possess terminal modifiable amine functional groups for
binding various targeting or guest molecules.14
PAMAM are
large, highly branched macromolecules that are synthesized
from an ethylenediamine core by a successive addition
of methyl acrylate and ethylenediamine. The number
of terminal amino groups increase as the generation of
PAMAM increases.15
The OPs, such as azinphos-methyl (AZM) and
methamidophos (MMP), are some of the most commonly
used pesticides in agriculture and domestic purposes.16-18
Its easy acquisition and handling can pose a great threat
if used as CW, and the potential attack employing these
agents gives a strong reason for continuous research on
the development of more effective antidotes against them.
Structure-based molecular characterization using
semi-empirical quantum mechanical studies19,20
can be
used to accurately understand the properties that govern
the interactions between molecular systems. However,
the application of these computational chemistry methods
to analyze nanostructured systems requires its suitable
adaptation and implementation.To solve the above problem,
a new discipline has emerged, called “nanoinformatics”,21
which meets all the advances in information technology,
nanotechnology and bioinformatics to be applied in
nanoscale research.22-24
In this work, a nanoinformatics methodology was used
to calculate the interaction energy between molecule(1)-
molecule(2) pairs.25,26
The first molecule represents the
monomeric unit of a specific dendrimer attached to a
functional group, and the second molecule represents the
OP compound. To obtain an accurate prediction of the
interaction energies, the nanoinformatics methodology
implements a Metropolis Monte Carlo algorithm27
that
performs a random sampling through Euler angles25
to
generate thousands of different molecular conformations
(for a specific pair), then for each new conformation, its
interaction energy is calculated using a semi-empirical
quantum mechanical method.19,20
The long list of
interaction energy values can be used for in silico analysis
of the affinity between OP agents and the functionalized
PAMAM. Thus, in silico techniques are perfect tools for
this task since they can quickly provide structures of several
new lethal compounds for further experimental work.28
The aim of the present work is to incorporate active
biomolecules such as folic acid, coumarin, lysine, arginine
and asparagine as new end-groups in the structures of
Table 1. OP compounds
Compound Use
Mammalian
toxicitya
Methamiphos
Insecticide 10-30
Azinphos-methyl
Insecticide 32
Diazinon
Insecticide 80-300
Dimethoathe
Insecticide 160-387
VX
CW 10
Soman
CW 35-50
Cyclosarin
CW 1.2
Sarin
CW 75-100
a
Mammalian toxicity for all CWAs is expressed in terms of the lethal
dose, whereas for pesticides, toxicity is expressed using the LD50 value
[concentration ingested (mg per kg of animal weight) at which half of
the tested animals die]. Because CWAs are highly volatile, most of the
deaths occurred by inhalation or skin contact, thus, values provided
indicate the lethal dose by inhalation (mg per min per m3
). All CWAs
have incapacitating effects that occur in 1-10 min and the lethal effect
occurs between 10-15 min.
3. Nano-Detoxification of Organophosphate Agents by PAMAM Derivatives J. Braz. Chem. Soc.582
PAMAM generation 4 and 5 (G4 and G5) to increase
the capacity to capture highly toxics OP compounds,
specifically, AZM and MMP.
Methodology
Materials
N-hydroxybenzotriazole (HOBt), 1-[3-(dimethylamino)
propyl]-3-ethylcarbodi-imide hydrochloride (EDC·HCl),
folic acid (FA), Boc-Arginine(Tos)-OH [Boc-Arg(Tos)-
OH], Boc-Lysine(Z)-OH [Boc-Lys(Z)-OH],
Boc-Asparagine(Trt)-OH [Boc-Asn(Trt)-OH], coumarin-
3-carboxylic acid (Cou), dimethyl sulfoxide (DMSO),
N,N-dimethylformamide (DMF) were purchased from
Sigma Aldrich Co. (Saint-Louis, MO, USA). Dialysis
membrane (M.W. cut-off of 500 Da) was purchased
from Spectrum Labs ADP. Methanol (HPLC grade) was
purchased from Merck (Germany). Dendrimers PAMAM
G4 and G5 were purchased from Dendritech, Inc. (USA).
Pure azinphos-methyl-[O,O-dimethyl-S-(3,4-dihydro-4-
keto-1,2,3-benzotriazinyl-3-methyl) dithiophosphate] and
methamidophos (O,S-dimethylphosphoramidothioate)
were purchased Sigma-Aldrich.
MALDI analysis
To confirm the molecular weight of surface modified
PAMAM, mass spectra analyses of the dendrimers were
performed on matrix-assisted laser desorption ionization-
time of flight (MALDI-TOF) equipment (BrukerAutoflex)
with a pulsed nitrogen laser (337 nm), operating in positive
ion reflector mode, using 19 kV acceleration voltage and
2,5-dihydroxybenzoic acid (DHB) as matrix.
Synthesis of PAMAM derivatives
Six different PAMAM formulations varying size and
surface charges were synthetized as described below.
PAMAM-G5-Folate derivative (G5-FA): conjugation
of G5 with FA was carried out by a condensation between
the γ-carboxyl group of FA and the primary amino group of
PAMAM, according to the previously reported method12,29
[available as Supplementary Information (SI)].
PAMAM-Coumarine derivative (G5-Cou): conjugation
of G5 with Cou was carried out by a condensation between
the carboxyl group of Cou and the primary amino group of
PAMAM, according to the previously reported method12,30
(available as SI).
PAMAM G4-Arginine(Tos)-OH (G4-Arg): conjugation
of G4 with Boc-Arg(Tos)-OH was carried out by a
condensation between the carboxyl group of arginine and
the primary amino group of PAMAM, according to the
previously reported method12,30
(available as SI).
PAMAM G4-Lysine(Z)-OH (G4-Lys): conjugation of
G4 with Boc-lys(Z)-OH was synthesized according to the
previously reported method12,30
(available as SI).
PAMAM G4-Asn(Trt)-OH) (G4-Asn): conjugation of
G4withBoc-Asn(Trt)-OHwascarriedoutbyacondensation
between the carboxyl group of Boc-asn(Trt)-OH and the
primary amino group of PAMAM, according to the
previously reported method12,30
(available as SI).
PAMAMG4-Arg(Tos)-OH/Lys(Z)-OH(PAMAMG4-Arg/
Lys): the functionalization of G4 with Boc-Arg(Tos)-OH and
Boc-Lys(Z)-OH,wassynthesizedaccordingtothepreviously
reported method12,30
(available as SI).
Affinity assays
For each PAMAM and functionalized PAMAM,
experiments were carried out to determine the extent
of binding and fractional binding of MMP in methanol
solutions. Briefly, functionalized PAMAM adjusted
at 5.83 × 10−3
mmol L−1
was mixed in a 1:1 v/v ratio
with a 0.07 mmol L−1
(10 ppm) of MMP to give a final
MMP/PAMAM mol L−1
ratio of 12:1. Assays were
performed at pH 4.5-5.5. The samples were mixed for
45 min with the PAMAM derivatives at constant room
temperature (25 °C), and then centrifuged at 10000 rpm
for 10 min. The concentrations of MMP in supernatants
separated from precipitated were analyzed by HPLC. The
adsorption efficiency of each OP compound by PAMAM
were evaluated by determining the percentage decrease
in the absorbance at each specific maximum absorbance
wavelength using the following equation:
( )
0
%
0
A A
Adsorption
A
−
= × 100 (1)
where A0 is the initial absorbance at specific wavelength
and A is the final absorbance at the same wavelength of
each OP.31
Chromatographic method
An Agilent 1260 Infinity HPLC system (USA) with
a photodiode array detector and a C-18 Lichrospher
100 RP-18 (250 mm × 4 mm i.d. × 5 µm) column was used
for the analysis of column eluents.
A 40 µL solution of the studied supernatant was
injected into the HPLC apparatus. An isocratic elution
with methanol-water mixture (70:30, v/v) at a flow rate of
4. Durán-Lara et al. 583Vol. 26, No. 3, 2015
1.5 mL min−1
was used as mobile phase. The eluents were
monitored at 230 nm by absorbance detection. For data
processing, the chromatographic software ChemStation
was used. The chromatographic determination was
performed at room temperature. Reproducibility of the
method was determined for individual standards. Three
replicates per pesticide and concentration level were
prepared.
Calibration procedures
AZM and MMP standards were prepared in methanol.
The concentrations ranged from 10, 5.0, 2.5 and 1.25 ppm.
Linear calibration curves were obtained by plotting the
peak areas of the individual chemicals as a function of
the concentration using the GraphPad Prism program
for Windows (GraphPad Software, Inc., San Diego, CA,
U.S.A.).
Statistical analysis
All the experiments were carried out in triplicate and the
student t-test was used to calculate the statistical differences
between the experimental compound and the negative
control. A p-value of < 0.05 was considered significant.
Nanoinformatics studies
Theoretical platform
Two OP compounds were chosen for detailed molecular
dynamic (MD) investigations, MMP and AZM. G4-Asn10,
which presented the highest binding capacity for MMP at
pH 7, was chosen for build systems and was parameterized
according to the rules for all-atom CHARMM27.32
This
force field was not included in conventional force field
due to the dendritic structures. Charges, missing bond,
angle, dihedral parameters of the PAMAM core, as well
as intermediate dendrons and the outer surface were
estimated from similar terms within the force field. An
atomistic model for G4-Asn10 was built at pH 7 containing
64 protonated amines in total (54 terminal amines from
PAMAM and 10 amines fromAsn end groups).The 10 units
of Asn functional groups have been homogeneously
distributed in 64 end groups. The molecular parameters
for MMP and AZM were obtained from SwissParam33
online server (http://www.swissparam.ch/) by using
the Merck Molecular Force Field (MMFF) at pH 7 (net
charge = 0 for both molecules). The G4-Asn10 structure
and MMP and AZM molecules were implemented for
two systems. The first system contained 50 molecules of
MMP and 1 molecule of PAMAM (MMP: PAMAM, 50:1)
and the second one contained 50 molecules of AZM and
1 molecule of PAMAM (AZM: PAMAM, 50:1) in order
to evaluate the interaction of MMP and AZM molecules
over the entire surface of the PAMAM. For these models,
G4-Asn10 was set to the origin of the coordinate system.
Subsequently, the PAMAM was solvated in a water periodic
box (104 × 107 × 101 Å3
) of TIP3 water molecules,34
then
the corresponding number of MMP and AZM molecules
were added in a random way along the water box (Figure 1).
In order to neutralize the system, 64 chloride ions were
added for the first and second system, respectively. Both
systems were minimized for 10,000 steps and equilibrated
through MD simulation. The MD simulations were
performed at 298 K in the isobaric-isothermal ensemble for
20 ns. The temperature was maintained with the Langevin
thermostat with a damping coefficient of 1 ps−1
whereas
the pressure (1 atm) was kept constant using the Langevin
piston method.35
Equations of motion were integrated
with a 2 fs time step. The van der Waals (vdW) cut-off
was set to 12 and the long-range electrostatic forces were
computed using the particle-mesh Ewald approach. The
MD simulation was performed using the NAMD2.931
program and CHARMM27 force field36
for standard ions
and water molecules. All analyses were done with VMD37
software. For the analysis of the molecular dynamics, the
data collected along the trajectories were used to calculate
the radius of gyration, Rg, of the PAMAM and different
concepts were applied such as radial pair distribution
function (RDF) of MMP andAZM molecules. Furthermore,
several scripts were implemented in order to get relevant
information from de MD, such as capture of MMP and
AZM within a distance of 4 Å with respect of any atom
of the PAMAM for the MMP/AZM-PAMAM systems,
the number of hydrogen bond interactions between the
PAMAM and the entrapped MMP and AZM molecules.
Building molecular structures: The molecular structures
of MMP and the representative units of six functionalized
PAMAM (monomer-FA, monomer-Cou, monomer-Arg,
monomer-Lysine, monomer-Asparagine and monomer-
Amine) were built using GaussView program.38
The
geometry of these molecules was optimized at density
functional theory (DFT) level using B3LYP method with
6-31G* as the basis set, which has been implemented in
Gaussian 03 package program.39
Calculation of interaction energies by nanoinformatic
methodologies: Metropolis Monte Carlo simulations27
were used to calculate the interaction energy between
conformational pairs [molecule(1)-molecule(2)], in which
the molecule(1) represents the monomeric unit of a specific
PAMAM attached to a functional group and the molecule(2)
represents the OP compound MMP.
5. Nano-Detoxification of Organophosphate Agents by PAMAM Derivatives J. Braz. Chem. Soc.584
The algorithm that generates the conformational
sampling includes excluded-volume constraint method.25
Procedures used to calculate the interaction energy were as
follows: (i) the mass centers of molecule(1) and molecule(2)
are placed near the origin of the Cartesian coordinates frame,
(ii) then, the new orientation of molecule(2) [in relation to
molecule(1)] is chosen according to a set of three Euler
angles (α, β, γ) obtained randomly, (iii) molecule(2) is then
translatedalongtherandomvector“n”untilthevdWsurfaces
of each molecule touch each other, (iv) after translation,
single-point energy (1SCF) for this specific molecular
conformation [molecule(1)-molecule(2)] is calculated using
asemi-empiricalquantummechanicalmethod(PM6-DH+)19
implemented in MOPAC2009™ packaged program, version
11.038L (LINUX),29
(v) the heat of formation (∆Hf) is
extracted of the previous result and also from its isolated
parts. Then, a supermolecular approach was used to obtain
directly the interaction energy (∆Eint) for molecule-molecule
pairs, as the difference between the energy of the complex
“molecule(1)-molecule(2)” and the sum of the energies of its
isolated parts. According to the approach, the Gibbs energy
molecule-molecule, molecule-polymer or polymer-polymer
is defined by equation 2.
∆G = ∆Eint = ∆H – T∆S (2)
The entropy in the polymer systems25
has been shown
to have a small contribution in the ∆G, thus the term T∆S
is negligible and equation 3 can be used.
∆G = ∆Eint = ∆H (3)
The above equation is conveniently used to estimate
the interaction energy with a standard quantum chemical
procedure. In order to solve the superposition error
caused by this approach in equation 3, a highly efficient
conformational sampling, according to what is described
by MD calculations, the ∆G of mixing polymer-molecule
depends on the specific theory or model used. Thus, it was
employed PM6-DH+ theory that is based on the Flory-
Huggins lattice approximation, which is the simplest and
most widely used to calculate the free energy of polymer-
molecule interactions, ∆G. A general expression through
Flory-Huggins theory for the ∆G of a binary system
(molecule-molecule, molecule-polymer, or polymer-
polymer) can be described as:
1 2
1 2 1 2
1 2
ΔG
ln ln
RT x x
φ φ
φ φ χφ φ= + + (4)
where ∆G is the free energy of polymer-molecule per
mole of lattice site, φ is the volume fraction, and x is
the chain length with each repeating unit defined as
occupying a lattice site. The Flory-Huggins χ parameter is
defined as:
1,2z E
RT
χ =
∆
(5)
where z is the coordination number of the model lattice,
and ∆Eint is the energy of formation of an unlike pair (∆El,2).
According to equation 5, the energy of a particular [1,2]
pair is defined as:
Figure 1. G4-Asn10 models on a box containing explicit water molecules and Cl−
ions. The dendrimer structures are shown by space-filling representation
with carbon, nitrogen and oxygen atoms shown in green, blue and red, respectively. The Asn end groups are shown in purple. The Cl−
is shown in brown.
The chemical structures of MMP and AZM are shown in detail in the center. The distribution of 50 of these molecules in the water box are shown as space-
filling representation with carbon, nitrogen, oxygen, phosphorous and sulfur atoms shown in gray, blue, red, orange and yellow, respectively.
6. Durán-Lara et al. 585Vol. 26, No. 3, 2015
∆E1,2 = E1,2 – (E1 + E2) (6)
Finally, assuming that ∆G = ∆Eint (equation 3), the ∆Eint
described in this work was defined as:
∆E1,2 = ∆Eint = ∆Hf(molecule1 – molecule2) – (∆Hf(molecule1) +
∆Hf(molecule2)) (7)
Thus, steps (i) through (v) are repeated until the
obtention of 100,000 different molecular configurations
and their corresponding interaction energies. Then,
from this interaction energies distribution, the average
“<∆E(molecule1 – molecule2)>” was calculated.
The molecule(1) is replaced for each one of the
six representative units of the functionalized PAMAM
(monomer-FA, monomer-Cou, monomer-Arg, monomer-
Lysine, monomer-Asparagine and monomer-Amine) and
the above calculations are performed again to finally obtain
six-interaction energy averages.
In order to compare and demonstrate the effectiveness
of the above methodology, conformations associated with
the best interaction energies for each of the 6 analyzed
complexes were selected, and energies calculated at DFT
level using B3LYP method with 6-31G (d,p) as basis set,
which has been implemented in Gaussian 03 package
program37. Finally, the interaction energies were calculated
based on the supermolecular approach using the equation 7.
AChE inhibition assay
We evaluated in vitro the AChE inhibitory activity
of net MMP and encapsulated MMP with G4-Asn and
G4Arg/Lys. The MMP was measured as follows: PAMAM
derivatives with MMP were incubated by 45 min on stirring,
(molar ratio of MMP:PAMAM = 12:1), after that the
AChE inhibitory activity of MMP/PAMAM was measured.
Additionally, the AChE inhibitory activity of PAMAM
derivatives alone was also evaluated to disregard anyAChE
inhibitory activity of PAMAM. The assay for measuring
AChE inhibitory activity was carried out according to
Ellman et al.40
and adapted to 96-well microtiter plates
by Lopez et al.41
and described by Gutierrez et al.42
The
enzyme was obtained from electric eel (C-3389, typeVI-S,
Sigma Chemical Co., St. Louis, MO). In brief, 50 µL of
the sample dissolved in phosphate buffer (8 mmol L−1
K2HPO4, 2.3 mmol L−1
NaH2PO4, 150 mmol L−1
NaCl,
and 0.05% Tween 20 at pH 7.6) and 50 µL of the AChE
solution (0.25 unit mL−1
) in the same phosphate buffer
were added to the wells. Then, the plates were incubated
for 30 min at room temperature. After the incubation time,
100 µL of the substrate solution [40 mmol L−1
Na2HPO4,
0.2 mmol L−1
5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB),
and 0.24 mmol L−1
acetylthiocholine iodide (ACTI) in
distilled water at pH 7.5] was added. The absorbance
was read in a Bio-Tek Instrument microplate reader at
405 nm after 3 min. The enzyme activity was calculated as
a percentage compared to a control using only the buffer
and enzyme solution. The compounds were assayed at
100 µg mL−1
, and the alkaloid galanthamine was used as
the reference compound. When the enzyme inhibition
was > 50% at 100 µg mL−1
, dilutions were performed to
determine the corresponding IC50 values. The IC50 values
were calculated by means of regression analysis from three
individual determinations.
Results and Discussion
Synthesis of PAMAM derivatives
FA and Cou were attached to the amine terminal
groups of PAMAM-G5 by EDC·HCl and HOBt coupling
reaction.12,39
An excess amount of reagents (1:140) was used
to allow functionalization of the 128-terminal amine groups
of the PAMAM-G5 (MW ca. 28826) in order to ensure the
maximum functionalization. However, only 18 FA (MW
of PAMAM G5 + 18 FA ca. 36800) and 67 Cou molecules
(MW of PAMAM G5 + 67 Cou ca. 41600) were found
attached to the G5, respectively. It is proposed that the
difference in the number of molecules coupled to G5 was
due to steric hindrance caused by the size of FA and Cou,
respectively (Figures 2a and 2b).
Lys, Arg and Asn were coupled to the terminal amines
of the G4 by EDC·HCl and HOBt coupling reaction.12,39
An
excess amount of reagent (1:70) was used in all three cases
to allow maximum possible functionalization. Nevertheless,
only 44 molecules of Lys were conjugated to PAMAM
(MW G4 + 44 Lys ca. 30500), 15 molecules of arginine
were conjugated to G4 (MW G4 + 15 Arg ca. 19800),
and 10 molecules of Asn were found attached to the G4
(MW G4 + 10Asn ca. 18500).Again, it is proposed that the
specific number of molecules attached to G4 is determined
by the steric hindrance and physicochemical behavior of
Arg, Lys and Asn, respectively (Figures 2c-e).
The surface functionalization of PAMAM-G4 with two
different amino acids (Arg and Lys) was also achieved.
In this case, a small amount of arginine (1:5) was used
to accommodate second amino acid lysine by subsequent
reaction. With this simple methodology, coupling of two
different amino acids on the surfaces of PAMAM-G4
was obtained. The goal of this approach was to increase
the capacity of trapping to the OP (synergy) by PAMAM
derivatives through more hydrogen bonds, weak interactions
7. Nano-Detoxification of Organophosphate Agents by PAMAM Derivatives J. Braz. Chem. Soc.586
such as vdW, hydrophobic bonds, and electrostatic
interactions.Thus, it was possible to conjugate 14 molecules
ofArg (MW ca. 18600) to G4, and additionally introduce 25
molecules of Lys (MW ca. 25200) (Figure 2f).
The adsorption behavior of OPs compounds
(specifically MMP by PAMAM derivatives) in model
solutions was studied through HPLC analysis, but the
desorption behavior was not performed. (Figure S1, SI
section). It was observed that the adsorption of MMP was
higher when it was exposed to G4-Arg/Lys and G4-Asn
achieving 45% and 64% of affinity, respectively (Figure 3).
This high affinity may be due to the small size of MMP and
multiple hydrogen bonds that occur between the tertiary
amine and phosphoryl group from MMP and secondary,
tertiary amines (interdendritic) and carbonyl groups of
PAMAM derivatives (Figure 4). Molecular dynamics
calculations can predict and evaluate the contribution
of other energies involved in the dendrimer-OP system.
In this system, the hydrophobic interaction strength is
obtained through the vdW energy. The variation of the
vdW energy for the MMP-PAMAM and AZM-PAMAM
systems, are shown in Figure S2 (SI section). The vdW
energy pattern do not change significantly with time, it
confirms that vdW interaction is less significant than the
hydrogen bonding interaction. Another point to consider
is that G4-Asn provides greater amount of carbonyl
groups to the system than other polymers studied here,
allowing the formation of more hydrogen bonds between
the PAMAM and the OP.
Bioinformatic studies
Six, three-dimensional atomic models for monomer-
end-group complexes were generated using the in silico
design. These six functionalized PAMAM representative
structures were used to estimate the PAMAM/OPs
interactions.
The average interaction energy calculated by the
nanoinformatic methodologies is depicted in Table 2.
The theoretical data was correlated with the experimental
percentage of affinity calculated by HPLC (Figure 5). For
the six representative units of the studied functionalized
PAMAM, the relative order of affinity by MMP was
predicted as: monomer-Asparagine > monomer-Lysine >
monomer-Folate > monomer-Coumarin > monomer-
Arginine > monomer-Amine. As shown in Table 2
and Figure 5b, interaction energies calculated through
DFT-6-31G method showed a better correlation (r2
= 0.995)
as expected, corroborating the data obtained at semi-
empirical level. Furthermore, comparing DFT and PM6-
FH+ correlations (r2
), it can be noted that semi-empirical
method gave an acceptable and highly predictive
Figure 2. MALDI-TOF analysis of (a) G5-Cou67; (b) G5-FA18; (c) G4-Lys44; (d) G4-Asn10 (e) G4-Arg15; and (f) G4-Lys25-Arg14.
Figure 3. Experimental assays indicating the percentage of PAMAM
derivatives affinity to MMP. As control, G4-NH2 and G5-NH2 were used.
8. Durán-Lara et al. 587Vol. 26, No. 3, 2015
correlation. Moreover, the data obtained by DFT showed
to be much more accurate than semi-empirical methods,
however, considering the efficiency and speed required
for filtering several of thousand conformations, the semi-
empirical method probed to be commendable.
According to the obtained results, the functionalized
PAMAM improved its ability to trap OP molecules; thus,
strategic changes of the functional groups on the surface
of PAMAM have proven to be relevant when defining their
interaction with molecules of interest.
Computational chemistry approaches through
nanoinformatics platforms have proved to be essential in
the estimation of the PAMAM-molecule interactions, due to
the speedy results, the low cost of in silico experiments (this
is particularly true for those scenarios where the handling
of toxic or otherwise hazardous materials is necessary) and
the ability to relate the theoretical values with experimental
data with high accuracy (Figure 5). To characterize the
G4-Asn10 models and their interaction with MMP and
AZM molecules at molecular level, we performed MD
simulations to subsequently make a structural analysis of
the interactions present in the system. The first analysis
performed was the radius of gyration (Rg) calculated as
a function of simulated time, as shown in Figure S3 (see
SI section). Furthermore, the mean radius of gyration (Rg)
was calculated including only the last 8 ns of the 20 ns
equilibration simulations, where appeared to take on a
steady value. For G4-Asn10 with MMP molecules, values
of 21.0 ± 0.2 Å were observed, while for G4-Asn10 with
AZM molecules, 21.6 ± 0.3 Å. The second analysis was
RDF, which represents the probability to find an atom in
a shell (dr) at the distance r, of another atom or molecule.
Figure S4 (SI section) shows RDF of G4-Asn10/MMP
(1:50) and G4-Asn10/AZM (1:50) systems. The RDF
shows a radius r = 4 Å as the distance from which the
highest number of MMP molecules are found, whereas
Figure 4. Representation view of the interaction between MMP and G4-Asn indicating the role of hydrogen bond in the capture.
Figure 5. Correlation between the experimental affinity values obtained by HPLC studies: (a) the interaction energy averages calculated by using semi-
empirical method (MOPAC); and (b) the interaction energies calculated by DFT (B3LYP/6-31G).
9. Nano-Detoxification of Organophosphate Agents by PAMAM Derivatives J. Braz. Chem. Soc.588
for AZM molecules, the radius was higher and lower its
probability, radius r = 6Å and r =10Å forAZM molecules.
Therefore, the MMP molecules are positioned at locations
closer to PAMAM than AZM molecules, allowing greater
interaction with the terminal groups of G4-Asn10. Distances
were calculated between phosphorus atoms (P) of MMP/
AZM molecules and nitrogen atoms (N) of terminal
amines and Asn functional groups. The representation of
hydrogen bonds between MMP/AZM and G4-Asn10 are
shown in Figure 6. The AZM molecule can only form
one hydrogen bond with the G4-Asn10 monomers, and
the majority of the contributions come from the hydroxyl
oxygen atom not associated with the phosphate group.
On the other hand, a MMP molecule can achieve two
hydrogen bonds with G4-Asn10, the hydroxyl oxygen
atom from phosphate group and hydrogen atoms from
amine group. Thus G4-Asn10 can produce more hydrogen
bonds in the presence of MMP molecules than AZM,
for instance, in the Figure S5 (SI section) is depicted the
number of hydrogen bonds as functions of time for MMP
andAZM molecules interacting with G4-Asn10. The higher
number of hydrogen bonds is generated in the interaction
Table 2. Values of the theoretical interaction energies (at semi empirical and DFT level) and experimental (%) affinities between MMP and the monomeric
unit of PAMAM attached to six different functional groups
Compound No. Molecule 1 Molecule 2
Semi empirical
interaction
energy / (kcal mol−1
)
DFT
interaction
energy / (kcal mol−1
)
Experimental
affinity / %
1 monomer-Asn MMP −1.88 −29.01 64
2 monomer-Lys MMP −1.56 12.22 18
3 monomer-FA MMP −1.52 17.85 14
4 monomer-Cou MMP −1.46 19.05 13
5 monomer-Arg MMP −1.40 22.57 11
6 monomer-Amine MMP −1.30 23.64 6
r2
= 0.908 r2
= 0.995
Figure 6. Intermolecular hydrogen bonds (yellow circles) for MMP with G4 (a) and G4-Asn10 (b), intermolecular hydrogen bonds (yellow circles) for
AZM with G4 (c) and G4-Asn10 (d).
10. Durán-Lara et al. 589Vol. 26, No. 3, 2015
between G4-Asn10 with MMP molecules. The capability
of entrapment of G4-Asn10 was calculated through the
number of molecules near the PAMAM at a distance of
4 Å or less (value obtained from the RDF analysis). The
number of MMP trapped byG4-Asn10 was higher thanAZM:
approximately 18-20 MMP versus 10-12 AZM molecules
in the last 4 ns of the 20 ns equilibration simulation, in the
Figure S6 (available as SI) is depicted the number of MMP
and AZM trapped by functionalized PAMAM. Thus, it was
rationalized that the entrapment of these molecules was
promotedprimarilybyhydrogenbondsformation,especially
by of the MMP molecules ability to form more hydrogen
bonds with G4-Asn10.
AChE inhibition assay of PAMAM derivatives
To give further support to our model studies for
capturing affinity and molecular simulations data, the best
PAMAM derivatives (G4-Asn and G4-Arg/Lys, according
to percentage of affinity) were employed to evaluate
the inhibition of the AChE activity in vitro. Initially,
the inhibition activity of MMP to AChE was measured,
obtaining an IC50 of 0.85 µg mL−1
, confirming the higher
toxicity of MMP. Next, MMP was exposed (incubated)
to PAMAM derivatives and G4-NH2 (commercial) for
a period of 45 min with constant stirring (molar ratio
MMP:PAMAM = 12:1). IC50 values for commercial
PAMAM, MMP and derivatized PAMAM are given in
Table 3. In the case of MMP trapped by G4-NH2, the
data obtained indicated that the IC50 increased slightly,
showing IC50 values of 4.23 µg mL−1
. Whereas the MMP
trapped by PAMAM derivatives reached IC50 values of
65 µg mL−1
and 132 µg mL−1
forG4-Asn and G4-Arg/Lys,
respectively. Therefore, the IC50 of MMP encapsulated
PAMAM derivatives was significantly higher than MMP
encapsulated PAMAM-NH2 and free MMP (see Table 3).
Finally, the toxicity of PAMAM derivatives was evaluated
(G4-Asn and G4-Arg-Lys). In both cases, the values were
higher than 300 µmol L−1
(see Table 3), demonstrating
that the toxicity of PAMAM is not significant. These data
indicate that PAMAM derivatives have the capacity to
capture MMP efficiently, and perhaps, could be used in a
near future in preventing the poisoning events. Molecular
simulation calculations performed in this study predict that
there is a good “hydrogen bond” formation and “radius of
gyration” between G4-Asn with MMP. This is consistent
with the AChE activity assay, where the IC50 of MMP
increased significantly when coupled with PAMAM
derivatives. We hypothesize it is due to a higher MMP-
PAMAM union, due to the formed hydrogen bonds and the
closeness of the atoms in each of the molecules involved in
the interaction system. Furthermore, if we analyze the in
vitro experimental data obtained in theAchE activity assay,
we could conclude that the interaction MMP-PAMAM
is strong, limiting the contact between MMP and AchE
enzyme, presumably by the encapsulation of MMP inside
of PAMAM.
Conclusion
Functionalization of PAMAM derivatives with different
amino acids such as lysine, arginine, asparagine and others
biomolecules (coumarin and folic acid) was demonstrated.
These PAMAM derivatives were employed to trap AZM
and MMP, OP compounds broadly used as pesticides in
the agricultural sector. The molecules were designed and
modeled using novel bioinformatic tools, and chemically
synthesized, (specifically G4-Asn and G4-Arg/Lys), to
demonstrate significant affinity for MMP. The results
obtained by HPLC in model solutions, support theoretical
chemistry through nanoinformatic methodologies to
estimate the interaction energies between MMP and six
functionalized PAMAM. In silico results have proven to
be highly predictive to estimate a priori, a tendency of
affinity from a group of MPP by specific PAMAM, and
allowed observing that the PAMAM-molecule interactions
play an important role for the development of chemical
encapsulation and attachment systems. Moreover, by
comparing theoretical affinity of PAMAM derivatives with
AZM and MMP, it was demonstrated that the better trapping
of MMP was produced by PAMAM-Asn primarily due to
hydrogen bonds formation, especially by the MMP ability
to form more hydrogen bonds with G4-Asn10. Furthermore,
our results of affinity by HPLC and molecular dynamics
correlated highly with the in vitro enzyme AChE activity
assays. The inhibition of AChE by MMP in solution,
Table 3. AChE activity assay with MMP, PAMAM derivatives and their
formulations
Compound
AchE
IC50
(µg mL−1
) (µmol L−1
)
MMP 0.85 ± 0.05 6 ± 0.35
MMP/G4 4.23 ± 0.1 30 ± 0.8
MMP/G4-Asna
65 ± 1 > 300
MMP/G4-Arg-Lysa
132 ± 2 > 300
G4-Asn > 300 > 300
G4-Arg-Lys > 300 > 300
Galantamine 1.1 3.0
a
Mass and concentration displayed is in relation to the MMP amount
encapsulated by PAMAM (MMP/PAMAM = 12:1). n = 3, p < 0.05.
11. Nano-Detoxification of Organophosphate Agents by PAMAM Derivatives J. Braz. Chem. Soc.590
highly decreased when G4-Arg/Lys and G4-Asn were used
compared with commercial PAMAM G4. This information
allows us to conclude that PAMAM derivatives G4-Arg/
Lys and G4-Asn are capable of capturing MMP in solution
efficiently, and greatly reduce the MMP action in the
enzyme and could be used as possible nano-detoxification
agents of OPs in the future. In vivo studies for nano-
detoxication of OPs are under current investigation.
Supplementary Information
Supplementary data are available free of charge at
http://jbcs.sbq.org.br as PDF file.
Acknowledgements
This work was supported by a grant from E. Durán
and L. S. Santos FONDECYT (Postdoctoral Grant No.
3120178) and Innova Chile CORFO Code FCR-CSB
09CEII-6991. The authors acknowledge support from
Proyecto Anillo Científico ACT1107. Fabian Avila thanks
for doctoral scholarship to CONICYT-PCHA/Doctorado
Nacional 2013-21130308. Fondecyt 11130086 (F.M.N.)
and PIEI (Química y Bio-orgánica en Productos Naturales)
from UTalca is also acknowledged.
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Submitted: September 9, 2014
Published online: February 3, 2015