Molecular Docking and Dynamics Simulation Studies Predict Potential Anti-ADAR2 Inhibitors: Implications for the Treatment of Cancer, Neurological, Immunological and Infectious Diseases.

Altered RNA editing has been linked to several neurodevelopmental disorders, including autism spectrum disorder (ASD) and intellectual disability, in addition to depression, schizophrenia, some cancers, viral infections and autoimmune disorders. The human ADAR2 is a potential therapeutic target for managing these various disorders due to its crucial role in adenosine to inosine editing. This study applied consensus scoring to rank potential ADAR2 inhibitors after performing molecular docking with AutoDock Vina and Glide (Maestro), using a library of 35,161 compounds obtained from traditional Chinese medicine. A total of 47 compounds were predicted to be good binders of the human ADAR2 and had insignificant toxicity concerns. Molecular dynamics (MD) simulations, including the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) procedure, also emphasized the binding of the shortlisted compounds. The potential compounds had plausible binding free energies ranging from -81.304 to -1068.26 kJ/mol from the MM/PBSA calculations. ZINC000085511995, a naphthoquinone had more negative binding free energy (-1068.26 kJ/mol) than inositol hexakisphosphate (IHP) [-873.873 kJ/mol], an agonist and a strong binder of ADAR2. The potential displacement of IHP by ZINC000085511995 in the IHP binding site of ADAR2 could be explored for possible deactivation of ADAR2. Bayesian-based biological activity prediction corroborates the neuropharmacological, antineoplastic and antiviral activity of the potential lead compounds. All the potential lead compounds, except ZINC000014612330 and ZINC000013462928, were predicted to be inhibitors of various deaminases. The potential lead compounds also had probability of activity (Pa) > 0.442 and probability of inactivity (Pi) < 0.116 values for treating acute neurologic disorders, except for ZINC000085996580 and ZINC000013462928. Pursuing these compounds for their anti-ADAR2 activities holds a promising future, especially against neurological disorders, some cancers and viral infections caused by RNA viruses. Molecular interaction, hydrogen bond and per-residue decomposition analyses predicted Arg400, Arg401, Lys519, Trp687, Glu689, and Lys690 as hot-spot residues in the ADAR2 IHP binding site. Most of the top compounds were observed to have naphthoquinone, indole, furanocoumarin or benzofuran moieties. Serotonin and tryptophan, which are beneficial in digestive regulation, improving sleep cycle and mood, are indole derivatives. These chemical series may have the potential to treat neurological disorders, prion diseases, some cancers, specific viral infections, metabolic disorders and eating disorders through the disruption of ADAR2 pathways. A total of nine potential lead compounds were shortlisted as plausible modulators of ADAR2.

Therapeutic Perspectives of Adenosine Deaminase Inhibition in Cardiovascular Diseases.

Adenosine deaminase (ADA) is an enzyme of purine metabolism that irreversibly converts adenosine to inosine or 2'deoxyadenosine to 2'deoxyinosine. ADA is active both inside the cell and on the cell surface where it was found to interact with membrane proteins, such as CD26 and adenosine receptors, forming ecto-ADA (eADA). In addition to adenosine uptake, the activity of eADA is an essential mechanism that terminates adenosine signaling. This is particularly important in cardiovascular system, where adenosine protects against endothelial dysfunction, vascular inflammation, or thrombosis. Besides enzymatic function, ADA protein mediates cell-to-cell interactions involved in lymphocyte co-stimulation or endothelial activation. Furthermore, alteration in ADA activity was demonstrated in many cardiovascular pathologies such as atherosclerosis, myocardial ischemia-reperfusion injury, hypertension, thrombosis, or diabetes. Modulation of ADA activity could be an important therapeutic target. This work provides a systematic review of ADA activity and anchoring inhibitors as well as summarizes the perspectives of their therapeutic use in cardiovascular pathologies associated with increased activity of ADA.

Identification of Adenosine Deaminase Inhibitors by Metal-binding Pharmacophore Screening.

Adenosine deaminase (ADA) is a human mononuclear Zn2+ metalloenzyme that converts adenosine to inosine. ADA is a validated drug target for cancer, but there has been little recent work on the development of new therapeutics against this enzyme. The lack of new advancements can be partially attributed to an absence of suitable assays for high-throughput screening (HTS) against ADA. To facilitate more rapid drug discovery efforts for this target, an in vitro assay was developed that utilizes the enzymatic conversion of a visibly emitting adenosine analogue to the corresponding fluorescent inosine analogue by ADA, which can be monitored via fluorescence intensity changes. Utilizing this assay, a library of ∼350 small molecules containing metal-binding pharmacophores (MBPs) was screened in an HTS format to identify new inhibitor scaffolds against ADA. This approach yielded a new metal-binding scaffold with a Ki value of 26±1 µM.

Repurposing of Streptomyces antibiotics as adenosine deaminase inhibitors by pharmacophore modeling, docking, molecular dynamics, and in vitro studies.

Adenosine deaminase (ADA) is an enzyme present in purine metabolic pathway. Its inhibitors are considered to be potent drug lead compounds against inflammatory and malignant diseases. This study aimed to test ADA inhibitory activity of some Streptomyces secondary metabolites by using computational and in vitro methods. The in silico screening of the inhibitory properties has been carried out using pharmacophore modeling, docking, and molecular dynamics studies. The in vitro validation of the selected antibiotics has been carried out by enzyme kinetics and fluorescent spectroscopic studies. The results indicated that novobiocin, an aminocoumarin antibiotic from Streptomyces niveus, has significant inhibition on ADA activity. Hence, the antibiotic can be used as a lead compound for the development of potential ADA inhibitors.

A rapid assay to screen adenosine deaminase inhibitors from Ligustri Lucidi Fructus against metabolism of cordycepin utilizing ultra-high-performance liquid chromatography-tandem mass spectrometry.

Cordycepin has recently received increased attention owing to its extensive pharmacological activity. Adenosine deaminase (ADA) is widely distributed in mammalian blood and tissues; as a result, cordycepin is quickly metabolized upon entering into the body and converted into the inactive metabolite 3'-deoxyinosine, thus limiting its activity when administered alone. We herein present a novel ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for screening ADA inhibitors against the metabolism of cordycepin. Cordycepin and 3'-deoxyinosine were chosen as substrate and product, respectively. A proper separation was achieved for all analytes within 3 min. 3'-Deoxyinosine was quantified in the presence or absence of potential ADA inhibitors to evaluate ADA activity. The assay can simultaneously determine substrate and product, with the endogenous substance and ADA inhibitors added not interfering in its activity. After optimizing the enzymatic incubation and UHPLC-MS/MS conditions, Km and Vmax values for ADA deamination of cordycepin were 95.18 ± 7.85 µm and 363.90 ± 12.16 µmol/min/unit, respectively. Oleanolic acid and ursolic acid from Ligustri Lucidi Fructus were chosen as ADA inhibitors with half maximal inhibitory concentration values of 21.82 ± 0.39 and 18.41 ± 0.14 µm, respectively. A non-competitive inhibition model was constructed and this assay can be used to screen other potential ADA inhibitors quickly and accurately.

A new mixed inhibitor of adenosine deaminase produced by endophytic Cochliobolus sp. from medicinal plant seeds.

Medicinal plants have been studied for potential endophytic interactions and numerous studies have provided evidence that seeds harbor diverse microbial communities, not only on their surfaces but also within the embryo. Adenosine deaminase (ADA) is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Therefore, in this study, 20 types of medicinal plant seeds were used to screen endophytic fungi with tissue homogenate and streak. In addition, 128 morphologically distinct endophyte strains were isolated and their ADA inhibitory activity determined by a spectrophotometric assay. The strain with the highest inhibitory activity was identified as Cochliobolus sp. Seven compounds were isolated from the strain using a chromatography method. Compound 3 showed the highest ADA inhibitory activity and was identified as 5-hydroxy-2-hydroxymethyl-4H-pyran-4-one, based on the results of 1H and 13C NMR spectroscopy. The results of molecular docking suggested that compound 3 binds to the active site and the nonspecific binding site of the ADA. Furthermore, we found that compound 3 is a mixed ADA inhibitor. These results indicate that endophytic strains are a promising source of ADA inhibitors and that compound 3 may be a superior source for use in the preparation of biologically active ADA inhibitor compounds used to treat cancer.

Biochemical and computational insights of adenosine deaminase inhibition by Epigallocatechin gallate.

Epigallocatechin gallate, a flavonoid from Camellia sinensis possess various pharmacological activities such as anticancer, antimicrobial and antioxidant etc. Adenosine deaminase, (ADA), is a key enzyme involved in the purine metabolism, the inhibitors of which is being considered as highly promising candidate for the development of anti-proliferative and anti-inflammatory drugs. In this work we studied adenosine deaminase inhibitory activity of epigallocatechin gallate by using biophysical and computational methods. The enzyme inhibition study result indicated that epigallocatechin gallate possess strong inhibitory activity on ADA. ITC study revealed the energetics of binding. Also the binding is confirmed by using fluorescence spectroscopy. The structural details of binding are obtained from molecular docking and MD simulation studies.

Reynoutria Japonica from Traditional Chinese Medicine: A Source of Competitive Adenosine Deaminase Inhibitors for Anticancer.

BACKGROUND: Adenosine deaminase (ADA) is an important enzyme in purine metabolism and is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Traditional Chinese Herbal Medicine (TCHM) is widely used alone or in combination with chemotherapy to treat cancer, due to its ability to deliver a broad variety of bioactive secondary metabolites as promising sources of novel organic natural agents. OBJECTIVE: In the present study, 29 varieties of medicinal plants were screened for the presence of ADA inhibitors. RESULTS: Extracts from Reynoutria japonica, Glycyrrhiza uralensis, Lithospermum erythrorhizon, Magnolia officinalis, Gardenia jasminoides, Stephania tetrandra, Commiphora myrrha, Raphanus sativus and Corydalis yanhusuo demonstrated strong ADA inhibition with rates greater than 50%. However, Reynoutria japonica possessed the highest ADA inhibitory activity at 95.26% and so was used in our study for isolating the ADA inhibitor to be further studied. Eight compounds were obtained and their structures were identified. The compound H1 had strong ADA inhibitory activity and was deduced to be emodin by 1H and 13C-NMR spectroscopic analysis with an IC50 of 0.629 mM. The molecular docking data showed that emodin could bind tightly to the active site of ADA. Our results demonstrated that emodin displayed a new biological activity which is ADA inhibitory activity with high cytotoxic activity against K562 leukemia cells. The bioactivity of cordycepin was significantly increased when used in combination with emodin. CONCLUSION: Emodin may represent a good candidate anti-cancer therapy and adenosine protective agent.

Nephroprotective Effect of Essential Oils from Ginger (Zingiber officinale) and Turmeric (Curcuma longa) Rhizomes against Cadmium-induced Nephrotoxicity in Rats.

Several studies have shown that cadmium (Cd) induces nephrotoxicity and many plant foods phytochemicals have been found useful but their possible mechanism of action still remains unexplored. Hence, this study aimed to investigate the nephroprotective effect of essential oils from Nigeria ginger and turmeric rhizomes in cadmium-treated rats by examining their effect on renal function biomarkers (creatinine, urea and BUN), inflammatory cytokines (IL-6, IL-10 and TNF-Alpha) and renal adenosine deaminase (ADA) activity. The result revealed that essential oils from ginger and turmeric rhizomes exert anti-inflammatory effect by preventing alterations of renal function markers and cytokines (IL-6, IL-10 and TNF-Alpha) levels in Cd-treated rats. In addition, the essential oils inhibited renal ADA activity in Cdtreated rats. In conclusion, inhibition of ADA activity and modulation of inflammatory cytokines could be suggested as the possible mechanism of action by which essential oils from ginger and turmeric rhizomes exert their nephroprotective activities.

Urtica dioica inhibits cell growth and induces apoptosis by targeting Ornithine decarboxylase and Adenosine deaminase as key regulatory enzymes in adenosine and polyamines homeostasis in human breast cancer cell lines.

Breast cancer is a heterogeneous and multifactorial disease with variable disease progression risk, and treatment response. Urtica dioica is a traditional herb used as an adjuvant therapeutic agent in cancer. In the present study, we have evaluated the effects of the aqueous extract of Urtica dioica on Adenosine deaminase (ADA) and Ornithine decarboxylase (ODC1) gene expression in MCF-7, MDA-MB-231, two breast cancer cell lines being estrogen receptor positive and estrogen receptor negative, respectively.  Cell lines were cultured in suitable media. After 24 h, different concentrations of the extract were added and after 72 h, ADA and ODC1 gene expression as well as BCL2 and BAX apoptotic genes were assessed by Taqman real time PCR assay. Cells viability was assessed by MTT assay, and apoptosis was also evaluated at cellular level. The intra and extracellular levels of ODC1 and ADA enzymes were evaluated by ELISA. Results showed differential expression of ADA and ODC1 genes in cancer cell lines. In MCF-7 cell line, the expression level of ADA was upregulated in a dose-dependent manner but its expression did not change in MDA-MB cell line. ODC1 expression was increased in both examined cell lines. Also, increased level of the apoptotic BAX/BCL-2 ratio was detected in MCF-7 cells. These results demonstrated that Urtica dioica induces apoptosis in breast cancer cells by influencing ODC1 and ADA genes expression, and estrogen receptors. The different responses observed with these cell lines could be due to the interaction of Urtica dioica as a phytoestrogen with the estrogen receptor.

Rapid, reliable, and sensitive detection of adenosine deaminase activity by UHPLC-Q-Orbitrap HRMS and its application to inhibitory activity evaluation of traditional Chinese medicines.

Adenosine deaminase (ADA), which is a key enzyme in the metabolism of purine nucleosides, plays important roles in diverse disorders, such as tuberculosis, diabetes, liver disorders, and cancer. Determination of the activities of ADA and its isoenzymes in body fluids has received considerable attention in the diagnosis and treatment of relative diseases. Ultraviolet spectroscopy with adenosine (AD) as a substrate is a classical approach for screening potential ADA inhibitors by measuring the decrease in substrate (AD) at 265 nm or increase in the product (inosine) at 248 nm. However, AD and inosine share a very close maximum absorption wavelength, and the reaction is uncertain and is frequently interfered by the background color of matrix compounds or plant extracts. Thus, the method usually yields false positive or negative results. In this study, a novel, rapid, sensitive, and accurate ultra-high-performance liquid chromatography-Q exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometric (UHPLC-Q-Orbitrap HRMS) method was developed for determining and screening ADA inhibitors by directly determining the deamination product of AD, inosine. A proper separation was achieved for inosine and chlormequat (internal standard) within 2 min via isocratic elution (0.1% formic acid:methanol = 85:15, v/v) at a flow rate of 0.3 mL min-1 on a Waters ACQUITY HSS T3 column (2.1 mm × 100 mm, 1.8 µm) following a simple precipitation of proteins. The intra- and inter-day precisions of the developed method were below 7.17% and 8.99%, respectively. The method exhibited advantages of small total reaction volume (60 µL), short running time (2 min), high sensitivity (lowest limit of quantification of 0.02 µM for inosine), and low cost (small enzyme consumption of 0.007 unit mL-1 for ADA and substrate of 3.74 µM for AD in individual inhibition), and no matrix effects (101.64%-107.12%). Stability results showed that all analytes were stable under the investigated conditions. The developed method was successfully applied to the detection of the inhibitory activity of ADA from traditional Chinese medicines.

Syzygium cumini seed extract ameliorates adenosine deaminase activity and biochemical parameters but does not alter insulin sensitivity and pancreas architecture in a short-term model of diabetes.

BACKGROUND: The effects of the aqueous seed extract of Syzygium cumini (ASc) in a short-term model of diabetes in rats are little explored. The present study was designed to evaluate the effect of the ASc on adenosine deaminase (ADA) activity and on biochemical and histopathological parameters in diabetic rats. METHODS: ASc (100 mg/kg) was administered for 21 days in control and streptozotocin (STZ)-induced (60 mg/kg) diabetic rats. ADA activity, lipoperoxidation (cerebral cortex, kidney, liver and pancreas) and biochemical (serum) and histopathological (pancreas) parameters were evaluated. RESULTS: The main findings in this short-term model of Diabetes mellitus (DM) were that the ASc (i) significantly reverted the increase of ADA activity in serum and kidney; (ii) ameliorated the lipoperoxidation in the cerebral cortex and pancreas of the diabetic group; (iii) demonstrated hypolipidemic and hypoglycemic properties and recovered the liver glycogen; and iv) prevented the HOMA-IR index increase in the diabetic group. Therefore, the ASc can be a positive factor for increasing the availability of substrates with significant protective actions, such as adenosine. Moreover, by maintaining glycogen and HOMA-IR levels, the extract could modulate the hyperglycemic state through the direct peripheral glucose uptake. CONCLUSIONS: Our data revealed that the short-term treatment with ASc has an important protective role under pathophysiological conditions caused by the early stage of DM. These results enhance our understanding of the effect of the ASc on the purinergic system in DM.

Bio-solid-phase extraction/tandem mass spectrometry for identification of bioactive compounds in mixtures.

We describe a two-step column-based bioassay method with tandem mass spectrometric detection for rapid identification of bioactive species in mixtures. The first step uses an immobilized enzyme reactor (IMER) column interfaced to an electrospray ionization mass spectrometer (ESI-MS) to identify mixtures containing bioactive compounds (i.e., enzyme inhibitors), while the second step uses bioselective solid-phase extraction (bioSPE) columns to isolate compounds from "hit" mixtures, which are then identified online by data-dependent ESI-MS. IMER columns were prepared by entrapment of adenosine deaminase (ADA) into sol-gel derived monolithic silica columns, and used to perform a primary IMER screen of mixtures prepared from a bioactive library, which resulted in four apparent hit compounds. Such columns did not provide sufficient binding site density to allow bioSPE, and thus a new column format was developed using ADA that was covalently immobilized to monolithic silica capillary columns, providing ∼500-fold more protein binding sites than were present in columns containing entrapped proteins. Using the covalently linked ADA columns, bioactive mixtures identified by IMER were infused until a maximum total ion current was achieved, followed by washing with a buffer to remove unbound compounds. A harsh wash with 3% acetic acid eluted the strongly bound ligands and the resulting peak triggered data dependent MS/MS to identify the ligand, showing that two of the apparent hits were true ADA inhibitors and demonstrating the ability of this method to rapidly identify bioactive compounds in mixtures.

A carbon nanotubes based fluorescent aptasensor for highly sensitive detection of adenosine deaminase activity and inhibitor screening in natural extracts.

A carbon nanotubes (CNTs) based fluorescent aptasensor was developed for adenosine deaminase (ADA) activity detection and inhibitor screening by using adenosine (AD) as the substrate. This sensing system consists of CNTs, AD, split anti-AD aptamer fragment and dye-labeled aptamer fragment. In the absence of ADA, two aptamer fragments bind simultaneously with AD to form an AD-aptamer complex. This AD-aptamer complex cannot adsorb onto CNTs, and has high fluorescence intensity. When ADA is introduced into this system, ADA can convert AD into inosine, which has not affinity to the split anti-AD aptamer fragment. Thus, the split anti-AD aptamer fragments were adsorbed onto CNTs via strong π-π stacking interactions, resulting in the quenching of the fluorescence of the dye-labeled aptamer fragment. The proposed aptasensor can detect ADA activity from 0.005 to 0.2U/mL with a low detection limit of 0.002 U/mL. Moreover, it has been also demonstrated that this CNTs-based fluorescence aptasensor is suitable for ADA inhibitor screening from traditional Chinese medicine (TCM). Considering the superior sensitivity and specificity, the proposed CNTs-based fluorescent aptasensor can be expected to provide a simple, cost-effective and sensitive platform for the detection of ADA activity and screening of potential drugs.

Standard pentostatin dose reductions in renal insufficiency are not adequate: selected patients with steroid-refractory acute graft-versus-host disease.

BACKGROUND AND OBJECTIVE: Pentostatin is an irreversible inhibitor of adenosine deaminase and has been used to prevent graft-versus-host disease (GVHD) and to treat both acute and chronic GVHD. Dose reduction equations for patients with renal insufficiency are based on few patients with limited pharmacokinetic and clinical results. This phase II study (NCT00201786) was conducted to assess pentostatin efficacy and infectious complications seen from our previous phase I study in steroid-refractory acute GVHD (aGVHD). PATIENTS AND METHODS: Hospitalized patients with steroid-refractory aGVHD were given pentostatin 1.5 mg/m(2)/day intravenously on days 1-3 of each 14-day cycle. Prior to each dose, dose modifications were based on Cockcroft-Gault estimated creatinine clearance (eCrCL) with 30-50 mL/min/1.73 m(2) leading to a 50 % dose reduction and eCrCL less than 30 mL/min/1.73 m(2) leading to study removal. Plasma pentostatin area under the concentration-time curve (AUC) and incidence of infectious complications were evaluated. RESULTS: Two of the eight patients treated demonstrated excessive pentostatin exposure as determined by measurement of AUC. One of these patients had renal impairment, whereas the other patient demonstrated borderline renal function. Despite dose reduction to 0.75 mg/m(2), AUCs were significantly increased compared to the other patients in this study. Seven of eight patients treated with pentostatin had cytomegalovirus (CMV) viremia after pentostatin treatment; however none developed proven CMV disease. CONCLUSION: A 50 % dose reduction in patients with eCrCL 30-50 mL/min/1.73 m(2) seems reasonable. However, the eCrCL should be interpreted with extreme caution in patients who are critically ill and/or with poor performance status. Renal function assessment based on the Cockcroft-Gault method could be significantly overestimated thus risking pentostatin overdosing. These results imply a need to closely monitor pentostatin exposure in patients with renal insufficiency.

Aqueous seed extract of Syzygium cumini inhibits the dipeptidyl peptidase IV and adenosine deaminase activities, but it does not change the CD26 expression in lymphocytes in vitro.

Syzygium cumini (Sc) have been intensively studied in the last years due its beneficial effects including anti-diabetic and anti-inflammatory potential. Thus, the aim of this study was to evaluate the effect of aqueous seed extract of Sc (ASc) in the activity of enzymes involved in lymphocyte functions. To perform this study, we isolated lymphocytes from healthy donors. Lymphocytes were exposed to 10, 30, and 100 mg/mL of ASc during 4 and 6 h and adenosine deaminase (ADA), dipeptidyl peptidase IV (DPP-IV), and acetylcholinesterase (AChE) activities as well as CD26 expression and cellular viability were evaluated. ASc inhibited the ADA and DPP-IV activities without alteration in the CD26 expression (DPP-IV protein). No alterations were observed in the AChE activity or in the cell viability. These results indicate that the inhibition of the DPP-IV and ADA activities was dependent on the time of exposition to ASc. We suggest that ASc exhibits immunomodulatory properties probably via the pathway of DPP-IV-ADA complex, contributing to the understanding of these proceedings in the purinergic signaling.

Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells.

hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure-activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.

Immobilized capillary adenosine deaminase microreactor for inhibitor screening in natural extracts by capillary electrophoresis.

A novel strategy for the preparation of in-column adenosine deaminase (ADA) microreactor and rapid screening of enzyme inhibitors in natural extracts was demonstrated. In this approach, ADA was encapsulated in anionic polyelectrolyte alginate that was immobilized on the surface of fused-silica capillary via ionic binding technique with cationic polyelectrolyte polyethylenimine (PEI). On-line enzyme inhibition study was performed by capillary electrophoresis (CE). The substrate and product were baselined separated within 75s. The enzyme activity was determined by the quantification of peak area of the product. Enzyme inhibition can be read out directly from the reduced peak area of the product in comparison with a reference electropherogram obtained in the absence of any inhibitor. The inhibition percentage was used to evaluate relative activity of ADA microreactor. A known ADA inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) was employed as a model compound for the validation of the inhibitor screening method, and the screening of ADA inhibitor in 19 traditional Chinese herbal medicines was performed.

Syzygium cumini inhibits adenosine deaminase activity and reduces glucose levels in hyperglycemic patients.

Syzigium cumini (L.) Skeels from the Myrtaceae family is among the most common medicinal plants used to treat diabetes in Brazil. Leaves, fruits, and barks of S. cumini have been used for their hypoglycemic activity. Adenosine deaminase (ADA) is an important enzyme that plays a relevant role in purine and DNA metabolism, immune responses, and peptidase activity. ADA is suggested to be an important enzyme for modulating the bioactivity of insulin, but its clinical significance in diabetes mellitus (DM) has not yet been proven. In this study, we examined the effect of aqueous leaf extracts of S. cumini (L.) (ASC) on ADA activity of hyperglycemic subjects and the activity of total ADA, and its isoenzymes in serum and erythrocytes. The present study indicates that: (i) the ADA activity in hyperglycemic serum was higher than normoglycemic serum and ADA activity was higher when the blood glucose level was more elevated; (ii) ASC (60-1000 microg/mL) in vitro caused a concentration-dependent inhibition of total ADA activity and a decrease in the blood glucose level in serum; (iii) ADA1 and 2 were reduced both in erythrocytes and in hyperglycemic serum. These results suggest that the decrease of ADA activity provoked by ASC may contribute to control adenosine levels and the antioxidant defense system of red cells and could be related to the complex ADA/DPP-IV-CD26 and the properties of dipeptidyl peptidase IV (DPP-IV) inhibitors which serve as important regulators of blood glucose.

Purinergic modulation in the development of the rat uncrossed retinotectal pathway.

Adenosine is a neuromodulator implicated in nervous system development and plasticity and its effects are mediated by inhibitory (A(1), A(3)) and excitatory (A(2a), A(2b)) receptors. The role of adenosine in the synaptic activity depends mainly on a balanced activation of A(1) and A(2a) receptors which are activated by various ranges of adenosine concentrations. Herein, we investigated the expression of A(1) and A(2a) receptors and also the accumulation of cAMP in the superior colliculus at different stages of development. Furthermore, we examined the effects of an acute in vivo blockade of adenosine deaminase during the critical period when the elimination of misplaced axons/terminals takes place with a simultaneous fine tuning of terminal arbors into appropriate terminal zones. Lister Hooded rats ranging from postnatal days (PND) 0-70 were used for ontogeny studies. Our results indicate that A(1) expression in the visual layers of the superior colliculus is higher until PND 28, while A(2a) expression increases after PND 28 in a complementary developmental pattern. Accordingly, the incubation of collicular slices with 5'-N-ethylcarboxamido-adenosine, a non-specific adenosine receptor agonist, showed a significant reduction in cAMP accumulation at PND 14 and an increase in adults. For the anatomical studies, the uncrossed retinotectal projections were traced after the intraocular injection of horseradish peroxidase. One group received daily injections of an adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl adenine), 10 mg/kg i.p.) between PND 10 and 13, while control groups were treated with vehicle injections (NaCl 0.9%, i.p.). We found that a short-term blockade of adenosine deaminase during the second postnatal week induced an expansion of retinotectal terminal fields in the rostrocaudal axis of the tectum. Taken together, the results suggest that a balance of purinergic A(1) and A(2a) receptors through cAMP signaling plays a pivotal role during the development of topographic order in the retinotectal pathway.