Activation Of Cellular Systems Research Articles

Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A

The normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2′-deoxy-5-fluorozebularine in place of the cytidine in the TC substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A’s preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.

Molecular Design, Synthesis, and Evaluation of SNIPER (ER) that Induces Targeted Protein Degradation of ERα.

Manipulation of protein stability using small molecules has a great potential for both basic research and clinical therapy. Based on our protein knockdown technology, we developed chimeric degrader molecules SNIPER(ER)s that target the estrogen receptor alpha (ERα) for degradation via the ubiquitin-proteasome system. This chapter describes the design and synthesis of SNIPER(ER) compounds and methods for the evaluation of their activity in cellular systems and in a tumor xenograft model.

Natural Nrf2 Activators from Juices, Wines, Coffee, and Cocoa

Juices, wine, coffee, and cocoa are rich sources of natural polyphenolic compounds that have potent antioxidant activities proven by in vitro and in vivo studies. These polyphenolic compounds quench reactive oxygen and nitrogen species (RONS) or reactive free radicals and act as natural antioxidants which are also able to protect against reactive oxygen species (ROS)-mediated oxidative damage, which elevates cellular antioxidant capacity to induce antioxidant defense mechanisms by modulating transcription factors. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcription factor encoded in humans. It is activated as a result of oxidative stress and induces the expression of its target genes. This is one of the most important cellular defense mechanisms against oxidative stress. However, the oxidative stress alone is not enough to activate Nrf2. Hence phytochemicals, especially polyphenolics, act as natural Nrf2 activators. Herein, this review discusses the natural products identified in juices, coffee, cocoa and wines that modulate Nrf2 activity in cellular systems.

Life span extension of model yeast Saccharomyces cerevisiae upon ethanol derived-clover bud extract treatment

Antioxidant properties of clove bud have been widely studied due to its potential use in the pharmaceuticals field. One of the chemical bioactive compounds that show antioxidant activity is flavonoid. Our study revealed that the flavonoid content of ethanol-derived extract of clove bud was approximately 93.245 mg QE/100 gram. Less is known regarding the mode of actions of antioxidant from clove bud in cellular systems. In this study, we used model organism yeast Saccharomyces cerevisiae to study the action of antioxidant activity in cellular systems. We found that ethanol-derived clove bud extract (100 ppm) enhanced cells viability following H2O2-induced oxidative stress. Interestingly, clove bud extract increased yeast-antioxidative stress tolerance phenotype in a dose-independent manner. Suggesting, prooxidant activity of clove bud extract. Mitochondria have been known to involve in oxidative stress tolerance mechanisms primarily via mitochondrial adaptive ROS-signaling. Our data revealed that yeast mitochondrial membrane potential was unchanged following 100 ppm extract treatment yet significantly increased in higher extract treatment. Our study indicated that 100 ppm extract-supplementation in yeast culture resulted in a higher survival rate of yeast after 15-days of incubation, compared to that without extract treatments. We suggest that clove bud extract (100 ppm) could enhance oxidative stress tolerance phenotype in yeast S. cerevisiae, which then attributed on life span extension through its ROS scavenging activity. Further study must be conducted to confirm the underlying mechanisms of clove bud extract both physiologically and genetically.

Modulation of UDP-glucuronidation by acridinone antitumor agents C-1305 and C-1311 in HepG2 and HT29 cell lines, despite slight impact in noncellular systems.

Among the studied antitumor acridinone derivatives developed in our laboratory, 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) and 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) exhibited cytotoxic and antitumor properties against several cancer types and were selected to be evaluated in preclinical and early-phase clinical trials. In the present work, we investigated the impact of C-1305 and C-1311 on UDP-glucuronosyltransferase (UGT) activity. Enzyme activity modulation was studied using HPLC by analyzing standard UGT substrate metabolism in the presence and absence of antitumor drugs. The investigations were performed in two model systems: (i) under noncellular conditions, including human liver microsomes (HLM) and recombinant UGT1A1, 1A9 and 1A10 isoenzymes and (ii) in tumor cells. There was observed a slight impact of studied drugs on enzyme activity. Only UGT1A1 action was altered by both compounds. The modulatory effects of UGT activity in cellular systems depended on the tumor cell type. In the case of HepG2, C-1305 and C-1311 strongly induced UGT activity, particularly for C-1311, at concentrations significantly lower than the EC50. This effect contradicted irinotecan mediated UGT inhibition. HT29 colon tumor cells were less sensitive than HepG2 to enzyme modulation in the presence of the studied compounds, particularly C-1305, where enzymatic inhibition similar to that of irinotecan was observed. The results demonstrated that UGT activity modulation should be expected in the case of antitumor therapy with C-1305 or/and C-1311. Analysis of the results indicated that these modulations would occur via cellular regulatory pathways not by direct drug-enzyme interactions.

Three-Component Aminoalkylations Yielding Dihydronaphthoxazine-Based Sirtuin Inhibitors: Scaffold Modification and Exploration of Space for Polar Side-Chains.

Nonpolar derivatives of heterocyclic aromatic screening hits like the non-selective sirtuin inhibitor splitomicin tend to be poorly soluble in biological fluids. Unlike sp3 -rich natural products, flat aromatic compounds are prone to stacking and often difficult to optimize into leads with activity in cellular systems. The aim of this work was to identify anchor points for the introduction of sp3 -rich fragments with polar functional groups into the newly discovered active (IC50 = 5 μM) but nonpolar scaffold 1,2-dihydro-3H-naphth[1,2-e][1,3]oxazine-3-thione by a molecular modeling approach. Docking studies were conducted with structural data from crystallized human SIRT2 enzyme. Subsequent evaluation of the in silico hypotheses through synthesis and biological evaluation of the designed structures was accomplished with the aim to discover new SIRT2 inhibitors with improved aqueous solubility. Derivatives of 8-bromo-1,2-dihydro-3H-naphth[1,2-e][1,3]oxazine-3-thione N-alkylated with a hydrophilic morpholino-alkyl chain at the thiocarbamate group intended for binding in the acetyl-lysine pocket of the enzyme appeared to be promising. Both the sulfur of the thiocarbamate and the bromo substituent were assumed to result in favorable hydrophobic interactions and the basic morpholino-nitrogen was predicted to build a hydrogen bond with the backbone Ile196. While the brominated scaffold showed moderately improved activity (IC50 = 1.8 μM), none of the new compounds displayed submicromolar activity. Synthesis and characterization of the new compounds are reported and the possible reasons for the outcome are discussed.

Molecular Design, Synthesis, and Evaluation of SNIPER(ER) That Induces Proteasomal Degradation of ERα.

Manipulation of protein stability using small molecules has a great potential for both basic research and clinical therapy. Based on our protein knockdown technology, we recently developed a novel small molecule SNIPER(ER) that targets the estrogen receptor alpha (ERα) for degradation via the ubiquitin-proteasome system. This chapter describes the design and synthesis of SNIPER(ER) compounds, and methods for the evaluation of their activity in cellular system.

Klotho Prevents Translocation of NFκB.

Klotho protein is a β-glucuronidase capable of hydrolyzing steroid β-glucuronides. Two molecules are produced by the Klotho gene, a membrane bound form and a circulating form. This protein is recognized as an antiaging gene with pleiotropic functions. The activation of cellular systems is associated with the pathogenesis of several chronic and degenerative diseases associated with an inflammatory state. Inflammation is characterized by an activation of NFκB. Klotho suppresses nuclear factor NFκB activation and the subsequent transcription of proinflammatory genes. This review focuses on the current understanding of Klotho protein function and its relationship with NFκB regulation, emphasizing its potential involvement in the pathophysiologic process.

Enhancing the cellular anti-proliferation activity of pyridazinones as c-met inhibitors using docking analysis

A series of 2, 6-disubstituted pyridazinone derivatives were evaluated and optimized for their c-Met inhibitory activity in enzyme and cellular assay. An analysis of the SAR results arising from computer modeling analysis of members of the library led to the proposal that in order to obtain optimal inhibitory activity in cellular systems the lipophilic/hydrophilic properties of individual structural fragments in the inhibitors need to match those of corresponding binding pockets in the enzyme. Guided by this proposal, the quinoline-pyridazinone 8a, containing hydrophobic 6-indolyl pyridazinone and quinoline moieties along with a hydrophilic morpholine terminal group, was designed and synthesized. The results of studies with this substance showed that it is a selective c-Met inhibitor with both a high enzyme inhibition IC50 value of 4.2 nM and a high EBC-1 cell proliferation inhibition IC50 value of 17 nM.

Assay to probe proteolytic processing of Notch by γ-secretase.

With the increasing appreciation of the role of Notch in development and disease, measuring its cleavage and signaling activity in cellular systems has become important. Here we describe a cell-based method to analyze the cleavage of Notch at the S3 site by γ-secretase. HEK cells are transfected with an N-terminal truncated and myc-labeled mNotchΔE construct which can be easily and quantitatively detected by western blotting.

Direct Activation of Human Phospholipase C by Its Well Known Inhibitor U73122

Phospholipase C (PLC) enzymes are an important family of regulatory proteins involved in numerous cellular functions, primarily through hydrolysis of the polar head group from inositol-containing membrane phospholipids. U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione), one of only a few small molecules reported to inhibit the activity of these enzymes, has been broadly applied as a pharmacological tool to implicate PLCs in diverse experimental phenotypes. The purpose of this study was to develop a better understanding of molecular interactions between U73122 and PLCs. Hence, the effects of U73122 on human PLCβ3 (hPLCβ3) were evaluated in a cell-free micellar system. Surprisingly, U73122 increased the activity of hPLCβ3 in a concentration- and time-dependent manner; up to an 8-fold increase in enzyme activity was observed with an EC50=13.6±5 μm. Activation of hPLCβ3 by U73122 required covalent modification of cysteines as evidenced by the observation that enzyme activation was attenuated by thiol-containing nucleophiles, l-cysteine and glutathione. Mass spectrometric analysis confirmed covalent reaction with U73122 at eight cysteines, although maximum activation was achieved without complete alkylation; the modified residues were identified by LC/MS/MS peptide sequencing. Interestingly, U73122 (10 μm) also activated hPLCγ1 (>10-fold) and hPLCβ2 (∼2-fold); PLCδ1 was neither activated nor inhibited. Therefore, in contrast to its reported inhibitory potential, U73122 failed to inhibit several purified PLCs. Most of these PLCs were directly activated by U73122, and a simple mechanism for the activation is proposed. These results strongly suggest a need to re-evaluate the use of U73122 as a general inhibitor of PLC isozymes.

Novel Bis-platinum Complexes Endowed with an Improved Pharmacological Profile

Multinuclear platinum complexes are characterized by a peculiar DNA binding mode and higher cytotoxic potency than the mononuclear complexes, and efficacy against a wide range of preclinical tumor models. To reduce the high irreversible plasma protein binding and improve the chemical and metabolic drug stability, novel bis-platinum complexes were designed starting from the parent compound CT-3610. The novel second-generation bis-platinum complexes utilize alkylcarboxylate as leaving groups to improve their pharmacokinetic and pharmacodynamic profiles, thus overcoming the limitations of the previously developed multinuclear compounds. The selected compounds [CT-47518 and CT-47463, respectively (bis-capronate) platinum and (bis-butyrate) platinum], have similar in vitro degradation kinetics in human and murine plasma and, above all, an increased stability when compared to CT-3610, particularly in human plasma. In addition, both compounds exhibited a marked cytotoxic potency as compared with cisplatin and oxaliplatin. Interestingly, they were capable of overcoming resistance mediated by DNA mismatch repair defects in different cellular models. The complexes showed marked antitumor efficacy in Pt-refractory tumor xenografts, with remarkable activity in terms of tumor growth inhibition and tumor growth delay. The improved stability profile in human plasma compared to early bis- and triplatinum complexes together with the marked activity in cellular systems as well as in in vivo models, make CT-47518 and CT-47463 attractive candidates for further development.

Anti-inflammatory activity of four Bolivian Baccharis species (Compositae)

Hexanic, dichloromethanic, ethanolic and aqueous extracts from Baccharis obtusifolia HBK, Baccharis latifolia (R. et P.) Pers., Baccharis pentlandii D.C. and Baccharis subulata Wedd., plants used in the traditional medicine of South America have been studied for their in vitro anti-inflammatory activity in cellular systems. Calcium ionophore A23187-stimulated mouse peritoneal macrophages were validated as a source of cyclooxygenase-1 (COX-1) (prostaglandin E 2, PGE 2) and 5-lipoxygenase (5-LOX) (leukotriene C 4, LTC 4), and mouse peritoneal macrophages stimulated with Escherichia coli lipopolysaccharide (LPS) were used for testing cyclooxygenase-2 (COX-2) (PGE 2), nitric oxide (NO) and tumour necrosis factor-α (TNF-α) activity. Most of the extracts tested were active in all assays.

Sesquiterpenes from Jasonia glutinosa: in vitro anti-inflammatory activity.

Four sesquiterpenes isolated from Jasonia glutinosa D.C. (Asteraceae), namely lucinone, glutinone, 5-epi-kutdtriol and kutdtriol, have been evaluated for their in vitro anti-inflammatory activity in cellular systems generating cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) metabolites. None of the compounds assayed had a significant effect on leukotriene C4 (LTC4)-release from calcium ionophore-stimulated mouse peritoneal cells. However, the release of prostaglandin E2 (PGE2) by mouse peritoneal cells stimulated with calcium ionophore was inhibited by these compounds, although with less potency than the reference drug indomethacin (IC50=0.24 microM). The IC50 values of the active compounds were: lucinone 42.69 microM, glutinone 3.61 microM, 5-epi-kutdtriol 1.28 microM and kutdtriol 39 microM. Of the tested compounds, only glutinone (IC50=24 microM) showed a significant effect on thromboxane B2 (TXB2)-release induced by calcium ionophore in human platelets, although with less potency than the reference drug ibuprofen (IC50=1.27 microM).

Formation of ARS-independent miniplasmids upon transformation of yeast Pichia methanolica with DNA molecules containing "transforming" and "nontransforming" genes

In the study of transforming DNA molecules in cells of the methylotrophic yeast Pichia methanolica, it was found that the efficiency of transformation and the possibility of autonomous replication of hybrid plasmids and miniplasmids consisting of only transforming gene (Trg) sequences do not depend on the presence of ARS sequences with the canonical structure, such as 2 microns DNA in the yeast Saccharomyces cerevisiae. Data supported a suggestion about the existence of the relationship between gene transforming activity, its replicative ability, and transcription. The efficiency of vector activity of Trg is not sufficient to provide the transformation of other sequences of transforming DNA (tDNA). The loss of nonselected sequences of tDNA observed upon transformation is the result of rearrangements and deletions and is associated with the activity of cellular systems involved in nucleic acid metabolism. Miniplasmids similar in structure were shown to differ in their stability in independent transformants. The rate of loss of autonomously replicating plasmids strongly varied (from 1 to 99%) under nonselective conditions and nearly did not depend on plasmid structure.

Effects of some iridoids from plant origin on arachidonic acid metabolism in cellular systems.

Seven iridoid glycosides isolated from different extracts of Scrophularia scorodonia L., namely bartsioside, aucubin, harpagide, harpagoside, 8-acetylharpagide, scorodioside and scropolioside B, had been evaluated for their in vitro anti-inflammatory activity in cellular systems generating COX and LOX metabolites. Structure-activity relationships obtained from in vitro screening results were discussed. Most compounds assayed did not exhibit any significant effect on PGE2- and LTC4-release from calcium ionophore-stimulated mouse peritoneal macrophages. In the LTC4-assay, only aucubin showed a significant effect, with an IC50 value of 72 microM. Harpagoside and harpagide also inhibited release of LTC4, but neither effect reached statistical significance. The release of PGE2 by mouse peritoneal macrophages stimulated with calcium ionophore was inhibited by harpagoside and 8-acetylharpagide, but this effect is not statistically significant. However, most iridoids assayed showed a significant effect on TXB2-release from calcium ionophorestimulated human platelets, with inhibition percentages slightly lower than the reference drug ibuprofen. Only harpagide, scorodioside and scropolioside B had no significant effect on TXB2-release. Our results indicate that selective inhibition of the TX-synthase enzyme may be the primary target of action of most of these iridoids, and one of the mechanisms through which they exert their anti-inflammatory effects.

Influence of non-nutrient plant components on biotransformation enzymes

Plant foods contain numerous non-nutritive substances which exert biological activity. Most attention has been focused on the anticarcinogenic effects of these compounds. Many of the mechanisms involved include induction or inhibition of biotransformation enzymes. Each individual has its own isoenzyme pattern for the various drug-metabolizing enzymes. The multiplicity of these enzymes results in differential responses to dietary constituents. A substance may increase the level of a certain P450, and decrease the level of another. Although this complicates matters considerably, it also offers the possibility of specifically influencing biotransformation directed at a particular compound, e.g., a cytostatic agent. Using the important class of the glutathione S-transferase (GST) as an example, the various phenotypic and genetic origins of interindividual variation are described. Genetic variation is especially important for the mu and thetra class enzymes. The induction of individual isoenzymes of the GST has been studied in man rat. It was shown that the changes in the GST isoenzyme pattern induced by Brussels sprouts in rat liver and intestine were very similar to that caused by administration of ally isothiocyanate, and not to that resulting from goitrin. In man Brussels sprouts led to induction of GST alpha only. A number of naturally occurring catechols, or more likely the quinones derived from them, are effective irreversible inhibitors of GST. Eugenol, for instance, lowers GST activity in man. A second class of compounds which shows promise are alpha, beta-unsaturated aldehydes and ketones. A number of naturally occurring representatives of this class inhibit GST pi irreversibly, and ethacrynic acid, a drug with a similar reactive moiety in its structure, has already been shown to be quite useful to inhibit GST activity in cellular systems. Several approaches for future studies on the effects of dietary constituents are indicated: 1) further studies on the mechanisms of induction and inhibition of biotransformation enzymes: 2) careful studies using human volunteers, where the effects can be studied in isolation as much as possible; 3) studies of the disposition and kinetics of the dietary constituents themselves, to assess the relevance of inducing agents in food for the day-to-day human situation.

Medical application of microcalorimetry in human toxicology. A study of blood compatibility of hemodialysis membranes

Abstract

Determination of cyclic nucleotide phosphodiesterase activity in cellular systems by ion-pair reversed-phase liquid chromatography

Determination of cyclic nucleotide phosphodiesterase activity in cellular systems by ion-pair reversed-phase liquid chromatography