Hemigramicidin-TEMPO conjugates: novel mitochondria-targeted anti-oxidants

Oxidative damage to various cellular constituents (such as, proteins and lipids) mediated by reactive oxygen species (ROS) is thought to be an important mechanism underlying the pathogenesis of a variety of acute and chronic diseases. Mitochondria are the main source of ROS within most cells. Accordingly, there is increasing interest in the development of pharmacological ROS scavengers, which are specifically targeted to and concentrated within mitochondria. Numerous compounds with these general characteristics have been synthesized and evaluated in a variety of in vitro and in vivo models of redox stress. Among the more promising of these mitochondria-targeted anti-oxidants are those that employ various peptides (or peptide-like moieties) derived from the antibiotic, gramicidin S, as the targeting construct and employ the stable free radical, 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-NH(2)-TEMPO), as the ROS scavenging "payload." One of these hemigramicidin-TEMPO conjugates, XJB-5-131, has been shown to ameliorate intestinal mucosal injury and prolong survival in rats subjected to lethal hemorrhage.     

Synthesis of 6-O-acyl-L-ascorbic acid-2-O-phosphates and study of their antioxidant effects in 95-D cells

Reactive oxygen species (ROS) are believed to play a role in development of several diseases. L-ascorbic acid (AsA) is a kind of excellent antioxidant, but its instability in solution and hydrophilicity limits the wide use of it. Structural modifications of AsAby the introduction of lipophilic moieties can lead to derivatives with increased stability against thermal and oxidative degradation. In this study, a series of 6-O-acyl-L-ascorbic acid-2-O-phosphates (6-Acyl-AA-2Ps) were synthesized from a stable AsA derivative, L-ascorbic acid-2-phosphate ester magnesium (AA-2P) and long-chain fatty acids, act as radical scavengers for ROS and free radicals. ROS scavenging ability was investigated by CDCFH method in 95-D cells. The effect of 6-Acyl-AA-2P series on viability of 95-D cells was also studied by MTT method. All the synthesized compounds show stronger ROS scavenging ability and cytotoxicity than those of AsA. High-performance liquid chromatography assay demonstrates that 6-Acyl-AA-2Ps are stable in RPMI-1640 medium and can easily permeate cell membrane and be converted into AsA and L-ascorbic acid-2-phosphate ester. The results also show that the activity of 6-Acyl-AA-2Ps increases with a decrease in the length of the alkyl chain but cytotoxicity decreases. The optimum length of alkyl side chain is 12 carbons. In summary, 6-Laur-AA-2P should be one of the best candidates for the development of an efficient new AsA derivative and should be further investigated in detail.     

Synthesis of 5-aryl-2H-tetrazoles, 5-aryl-2H-tetrazole-2-acetic acids, and [(4-phenyl-5-aryl-4H-1,2,4-triazol-3-yl)thio]acetic acids as possible superoxide scavengers and antiinflammatory agents

A series of 5-aryl-2H-tetrazoles, 5-aryl-2H-tetrazole-2-acetic acids, and [(4-phenyl-5-aryl-4H-1,2,4-triazol-3-yl)thio]acetic acids were synthesized and tested in vitro for superoxide scavenging activity, in vivo in the carrageenan-induced rat paw edema assay, and in the reverse passive Arthus reaction. The hydroxy-substituted compounds were effective as in vitro scavengers of superoxide but were not effective as in vivo antiinflammatory agents.     

Salen-manganese complexes as catalytic scavengers of hydrogen peroxide and cytoprotective agents: structure-activity relationship studies

Synthetic catalytic scavengers of reactive oxygen species (ROS) may have broad clinical applicability. In previous papers, two salen-manganese complexes, EUK-8 and EUK-134, had superoxide dismutase (SOD) and catalase activities and prevented ROS-associated tissue injury. This study describes two series of salen-manganese complexes, comparing catalytic ROS scavenging properties and cytoprotective activities. The compounds vary widely in ability to scavenge hydrogen peroxide, with this activity most influenced by salen ring alkoxy substitution and aromatic bridge modifications. In contrast, all compounds show comparable SOD activities. The most active alkoxy-substituted catalase mimetics protected cultured cells from hydrogen peroxide, and a subset of these were also neuroprotective in a rodent stroke model. Thus, structural modification of the prototype EUK-8 yields compounds with enhanced catalase activity and, in turn, biological effectiveness. This supports the concept that salen-manganese complexes represent a class of SOD and, in particular, catalase mimetics potentially useful against ROS-associated diseases.     

A simple assay for the measurement of plasma antioxidant status using spontaneous autoxidation of homovanillic acid

INTRODUCTION: Reactive oxygen species (ROS) contribute to the development of pathophysiological processes, hence the increasing interest in modulating the antioxidant status of patient by nutritional or pharmacological intervention. Antioxidants act by preventing the formation of ROS (inhibitory effect) and/or by trapping these species (scavenger effect). We have developed a simple, sensitive, and reliable test to measure the total antioxidative efficiency of plasma or other biological fluids using microliter samples. METHODS: Autoxidation of homovanillic acid (HVA) gives rise to fluorescent dimers. Antioxidants contained in the plasma (or free aqueous solutions) scavenge the ROS involved in this process and transiently stop the linear increase in fluorescence intensity during a time (delay) proportional to the total concentration of antioxidants and their scavenging efficiency. In addition to this scavenging effect, the kinetics of HVA autoxidation, restarting after the delay, reflects the ability of the plasma antioxidants to inhibit the ROS-triggered autoxidation. RESULTS: The rate of the HVA autoxidation depended on the temperature, the protonation of the phenolic group, and on the presence of peroxide, peroxyl radicals, and peroxidase as well as metal ions. This Fenton-like reaction was transiently stopped by various ROS scavengers including quercetin, ascorbic acid, and thiol derivatives (glutathione and N-acetylcystein) while metal chelating agents such as desferrioxamine, ethylene diamine tetracetic acid (EDTA), and polyamine only reduced its rate. DISCUSSION: The main advantages of this new assay are its versatility to investigate in a single run both the scavenging and inhibitory components of the antioxidant capacity, and its relevance to the reactive hydroxyl radical. As shown in this study, the increase in the antioxidant capacity of human plasma during pharmacological supplementation with antioxidant illustrates one of the various fields of application of this assay.     

Cellular HTS assays for pharmacological characterization of Na(V)1.7 modulators

Ion channels are challenging targets in the early phases of the drug discovery process, especially because of the lack of technologies available to screen large numbers of compounds in functionally relevant assays. The electrophysiological patch-clamp technique, which is the gold standard for studying ion channels, has low throughput and is not amenable to screening large numbers of compounds. However, for random high-throughput screening (HTS) of compounds against ion channel targets, a number of functional cellular assays have become available during the last few years. Here we use the sodium channel NaV1.7 stably expressed in human embryonic kidney 293 cells and compare three HTS assays-a Li flux atomic absorption spectroscopy (AAS) assay, a fluorescent imaging plate reader (FLIP, Molecular Devices, Sunnyvale, CA) membrane potential assay, and a fluorescence resonance energy transfer (FRET)-based membrane potential assay-to an automated electrophysiological assay (the Ionworks HT [Molecular Devices] platform) and characterize 11 known NaV inhibitors. Our results show that all three HTS assays are suitable for identification of NaV1.7 inhibitors, but as an HTS assay the Li-AAS assay is more robust with higher Z' values than the FLIPR and FRET-based membrane potential assays. Furthermore, there was a better correlation between the Ionworks assay and the Li-AAS assay regarding the potency of the NaV inhibitors investigated. This paper describes the first comparison between all the HTS assays available today to study voltage-gated NaVs, and the results suggest that the Li-AAS assay is more suited as a first HTS assay when starting an NaV drug discovery campaign.     

Development of fluorometric reactive oxygen species assay for photosafety evaluation

The present investigation involved an attempt to develop a new reactive oxygen species (ROS) assay system for the photosafety assessment of chemicals using 1,3-diphenylisobenzofuran (DPBF), a fluorescent probe for monitoring ROS generation. The assay conditions of the fluorometric ROS (fROS) assay were optimized focusing on the solvent system, concentration of DPBF, fluorescent determination, screening run time and reproducibility. The photoreactivity of 21 phototoxic and 11 non-phototoxic compounds was assessed by fROS assay, and the obtained ROS data were compared with the results from a micellar ROS (mROS) assay and in vitro/in vivo phototoxicity information to confirm the predictive capacity of the fROS assay. In the optimized fROS assay, intra-day and inter-day precision levels (coefficient of variation) were found to be below 5%, and the Z'-factor for DPBF fluorescence quenching showed a large separation between positive and negative controls. Of all tested compounds, 3 false positive and 7 false negative predictions were observed in the fROS assay, and the negative predictivity for the fROS assay was found to be lower than that for the mROS assay. Although the fROS assay has some limitations, the procedures for it were highly simplified with a marked reduction in screening run time and one analytical sample for monitoring ROS generation from compounds. The fROS assay has the potential to become a new tool for photosafety assessment at an early stage of product development.     

Discovery of a novel competitive inhibitor of PTP1B by high-throughput screening

AIM: The aim of the present study was to discover novel protein tyrosine phosphatase 1B (PTP1B) inhibitors. We expressed and purified the human PTP1B catalytic domain and set up a molecular level high-throughput screening (HTS) assay to screen a set of 48,000 pure compounds. RESULTS: HTS was finished with an averaged Zo factor of 0.63, and LGH00081, a competitive inhibitor of PTP1B with novel structure and relatively good selectivity for receptor-type protein tyrosine phosphatases, was identified. CONCLUSION: We established a molecular level assay which is useful for the screening of PTP1B inhibitors with therapeutic potential. The novel competitive PTP1B inhibitor LGH00081 offers a good start for structure modification and cellular functional activity study.     

Evaluation of the antioxidative properties of lipoxygenase inhibitors

BackgroundOxidative stress is a component of many pathological conditions including neurodegenerative diseases and inflammation. An important source of reactive oxygen species (ROS) are lipoxygenases (LOX) – enzymes responsible for the metabolism of arachidonic acid and other polyunsaturated fatty acids. LOX inhibitors have a protective effect in inflammatory diseases and in neurodegenerative disorders because of their anti-inflammatory activity. However, the molecular mechanism of the protective action of LOX inhibitors has not yet been fully elucidated.MethodsThe aim of this study was to compare the antioxidative potential of widely used LOX inhibitors: BWB70C, AA-861, zileuton, baicalein and NDGA. The antioxidative properties were evaluated in cell-free systems. We measured the effect of the tested compounds on iron/ascorbate-induced lipid peroxidation and on carbonyl group formation in the rat brain homogenate. Direct free radical scavenging was analyzed by using DPPH assay.ResultsOur data showed that the inhibitor of all LOXs, i.e., NDGA, 5-LOX inhibitor BWB70C and the inhibitor of 12/15-LOX, baicalein, significantly decreased the level of lipid and protein oxidation. The free radical scavenging activity of these inhibitors was comparable to known ROS scavengers, i.e., resveratrol and trolox. Zileuton (the inhibitor of 5-LOX) slightly prevented lipid and protein oxidation, it also scavenged the DPPH radical. AA-861 (the inhibitor of 5 and 12/15-LOX) slightly protected lipids against Fe/asc-evoked lipid peroxidation at high concentrations, but had no effect on carbonyl group formation and DPPH scavenging.ConclusionsOur results indicate that some LOX inhibitors demonstrate potent anti-oxidative, free radical scavenging properties. AA-861, whose antioxidative potential is very weak, may be a specific tool to be used in experimental and perhaps even clinical applications.     

氧化还原状态的改变控制手霉素诱导HL-60白血病细胞凋亡

目的研究手霉素对HL-60白血病细胞的作用,探讨其作用机制,主要是线粒体的改变。方法使用人的白血病细胞株HL-60细胞。MTT细胞毒性测定评价对白血病细胞的作用,使用Annexin V和一氧化氮(nitric oxide,NO)染料标记细胞,应用流式细胞仪术检测细胞内NO生成和细胞凋亡。细胞内超氧阴离子通过二氢乙啶(dihydroethidium,DHE)测定。应用化学发光法测定超氧歧化酶(superoxide dismutase,SOD)活性。采用荧光法测定谷胱甘肽(glutathi-one,GSH),萤光素-萤光素酶发光法测定ATP含量。免疫印迹技术检测细胞色素C和Mn-SOD的表达。结果手霉素引起HL-60细胞活性的下降,且呈剂量依赖性。手霉素诱导产生反应性氧基(reactive oxygen species,ROS):NO和超氧阴离子,降低GSH,但不影响SOD。手霉素诱导线粒体降低细胞ATP的含量,线粒体肿胀和细胞色素C从线粒体释放到细胞质。手霉素诱导的凋亡与ROS的增加有关。用N-乙酰基-L-半胱氨酸(N-acetyl-L-cysteine,NAC)抑制ROS可保护HL-60细胞逃避手霉素的细胞毒作用和避免手霉素诱导的凋亡。结论细胞内ROS的产生对手霉素的细胞毒作用起非常重要的作用。手霉素通过包括上游ROS产生,线粒体形态改变和细胞色素C释放的线粒体途径,诱导白血病细胞凋亡。     

High throughput screening assay for UDP-glucuronosyltransferase 1A1 glucuronidation profiling

Development of high throughput screening (HTS) assays for evaluation of a compound's toxicity and potential for drug-drug interactions is a critical step towards production of better drug candidates and cost reduction in the drug development process. HTS assays for drug metabolism mediated by cytochrome P450s are now routinely used in compound library characterization and for computer modeling studies. However, development and application of HTS assays involving UDP-glucuronosyltransferases (UGTs) are lagging behind. Here we describe the development of a fluorescence-based HTS assay for UGT1A1 using recombinant enzyme and fluorescent substrate in the presence of an aqueous solution of PreserveX-QML (QBI Life Sciences, Madison, WI) polymeric micelles, acting as a stabilizer and a blocker of nonspecific interactions. The data include assay characteristics in 384-well plate format obtained with robotic liquid handling equipment and structures of hits (assay modifiers) obtained from the screening of a small molecule library at the University of Wisconsin HTS screening facility. The application of the assay for predicting UGT-related drug-drug interactions and building pharmacophore models, as well as the effects of polymeric micelles on the assay performance and compound promiscuity, is discussed.     

Idiopathic chronic facial pain: Tricyclic antidepressant drug action is not due to free radical scavenging (antioxidant) activity

It has recently been suggested that free radicals (oxidants) are involved in stress-related conditions such as chronic idiopathic (psychogenic) facial pain. Tricyclic antidepressants are the drugs of choice in the treatment of this condition. This raises the question as to whether these compounds are acting as antioxidants (free radical scavengers). In this study the antioxidant properties of three tricyclic agents: amitriptyline, nortriptyline and dothiepin have been examined using two distinct assay systems. These involved testing scavenging activity against hypochlorous acid or against the peroxyl radical. None of the compounds tested showed any antioxidant/radical scavenging activity. Our results indicate that the therapeutic activity of these tricyclic antidepressants is not dependent on their ability to function as antioxidants.     

线粒体是双乙酰丙酮氧钒在肾上皮细胞中诱导产生活性氧物种的主要来源(英文)

本研究利用具有降糖效应的双乙酰丙酮氧钒确定其在两种肾上皮细胞系LLC-PK1和MDCK中诱导产生活性氧物种的来源。利用四种常用的荧光试剂分别检测了VO(acac)2诱导产生的过氧化氢(H2O2)和超氧阴离子(·O2)的水平并确定了它们在细胞内的主要来源部位。实验结果表明,VO(acac)2在LLC-PK1和MDCK两种肾细胞系中均能显著诱导ROS的生成,并且ROS主要来源于线粒体。本研究结果提示,可通过局部降低线粒体部位ROS的水平来减少钒化合物的毒性损伤,同时不影响钒化合物的活性。     

Promyelocytic HL60 cells express NADPH oxidase and are excellent targets in a rapid spectrophotometric microplate assay for extracellular superoxide.

A great number of drugs, toxicants, and growth factors induce the generation of intermediary reactive oxygen species (ROS). The human promyelocytic leukemia HL60 cell line differentiated along the macrophage or neutrophil lineage is a model system that is frequently used for the generation of ROS by various agents. As a primary source of ROS the superoxide anion produced by an enzymatic complex, NADPH oxidase, is well established. The present study shows that nondifferentiated HL60 cells contain NADPH oxidase and can be used as a model for the assessment of oxidant as well as antioxidant compounds. The expression of the multicomponent NADPH oxidase was demonstrated in nondifferentiated HL60 cells at the molecular level by detection of the mRNAs of the components gp91phox, p47phox, and p67phox as well as functionally by phorbol 12-myristate-13-acetate (PMA)-stimulated generation of superoxide, which was susceptible to inhibition by diphenyleneiodonium. The functional assay was performed using the cells in a log growth phase by adapting a standard microplate assay based on the classic superoxide dismutase-inhibitable reduction of cytochrome c. Validation of the microplate assay was carried out both with nonadherent differentiated HL60 cells and the adherent mouse monocyte-macrophage-like RAW 264.7 cell line, as well as with various compounds of oxidant (bleomycin sulfate, cis-diammineplatinum(II), camptothecin, TNF-alpha, IL-1 beta), nonoxidant (4 alpha-PMA, piracetam), and antioxidant (alpha-tocopherol, ascorbic acid) activity. In summary, we established a highly specific, reproducible and--with the aid of the nondifferentiated HL60 cell line--time-saving superoxide microplate assay as a valuable tool for the rapid screening of compounds for oxidative and antioxidative activity.     

High throughput pharmacology for drug discovery

High Throughput Screening (HTS) now plays an important role in the discovery of new lead compounds for novel therapeutic targets. The advantage of HTS over the conventional method, now termed as Low Throughput Screening (LTS), is that valuable compounds can be selected rapidly from a large number of samples with minimal human involvement. In spite of the growing awareness of HTS, the importance of the LTS in the drug discovery and development is still not changed. Advances in pharmacogenomics will also provide us many pharmacological targets, and thus increase the number of compounds that should be assayed by HTS and LTS. In this review, we will first describe the outline of HTS. We will next describe new approaches to develop and brush up the LTS: 1) screening method of drugs acting on ion channels by voltage-sensitive fluorescent dye, 2) functional assay method using reconstituted smooth muscle fiber, and 3) organ culture method as a useful model of vascular proliferative disease. These approaches, which work cooperatively with HTS, will contribute greatly to the development of new drugs.     

High-throughput screening with quantitation of ATP consumption: a universal non-radioisotope, homogeneous assay for protein kinase

A number of assays have been developed for high-throughput screening (HTS) of potentially bioactive compounds. To screen millions of chemical compounds efficiently, the best detection technology prior to initiating HTS must be chosen. Ideally, a non-radioisotope (non-RI), homogeneous method, equivalent to the most reliable assay for a particular target, should be selected as an HTS method. Protein kinases are among the most important classes for drug discovery because they participate in various signaling pathways. Several HTS technologies are available for kinase activity: SPA (Amersham, Piscataway, NJ, U.S.A.), HTRF (CIS-US, Inc., Bedford, MA, U.S.A.), IMAP (Molecular Devices, Sunnyvale, CA, U.S.A.), and Z'-LYTE (Invitrogen, Carlsbad, CA, U.S.A.). The amount of phosphorylated product is detected by different methods in these assays. Recently, Kinase-Glo Luminescent Kinase Assay, a non-RI, homogeneous, adenosine triphosphate (ATP) quantitative kit useful for kinase activity detection, has become available from Promega (Madison, WI, U.S.A.). ATP is a universal substrate for kinases. Thus, the Kinase-Glo assay shows promise for becoming the primary method of determining kinase activity in HTS. We have developed a Kinase-Glo system for cyclin-dependent kinase 4 (Cdk4), and compare its results with those of the filtration method, the most reliable assay for in vitro Cdk4 activity. In addition, the reliability and sensitivity of the Kinase-Glo are discussed.     

A robust homogeneous binding assay for α4β2 nicotinic acetylcholine receptor

AIM: To develop a homogeneous high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel alpha4beta2 nicotinic acetylcholine receptor (nAChR) modulators. METHODS: Membrane preparation of HEK293 cells expressing alpha4beta2 nAChR, [(3)H]cytisine and wheat germ agglutinin (WGA)-coupled microbeads were used to develop an HTS assay based on SPA technology. This method was validated against a conventional filter binding approach and applied to large-scale screening of a library containing 32 000 synthetic compounds. Intracellular calcium measurement was carried out to verify the bioactivities of the hits found by the SPA assay. RESULTS: IC(50) values of 2 reference compounds (epibatidine and RJR 2403) determined by SPA and filter binding methods were comparable and consistent with those reported elsewhere. A total of 54 compounds, showing more than 60% competitive inhibition on [(3)H]cytisine binding to alpha4beta2 nAChR, were identified initially following an HTS campaign. Secondary screening confirmed that 17 compounds with novel chemical structures possessed relatively high binding affinity to alpha4beta2 nAChR (K(i)<2 micromol/L). Eight compounds displayed antagonistic effects with >50% inhibition on ABT-594-induced calcium mobilization while none showed any agonist activity. CONCLUSIONS: This homogeneous binding assay is a highly efficient, amenable to automation and robust tool to screen potential alpha4beta2 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels.     

Mitochondrial defects and cytotoxicity by antimycin A on cultured osteoblastic MC3T3-E1 cells

Antimycin A (AMA), which inhibits complex III of the electron transport system, has been used as a reactive oxygen species (ROS) generator in biological systems. We investigated the effects of AMA on various parameters related to mitochondrial function in osteoblastic MC3T3-E1 cells. Here, we show that AMA-induced cell death was accompanied by the loss of ATP, complex I and IV activities, and mitochondrial membrane potential. Moreover, AMA stimulated oxidative stress and induced cytochrome c release from mitochondria in osteoblasts. Our data support AMA-induced death in osteoblasts via a mitochondria-dependent pathway. These biochemical changes in mitochondria were effectively prevented upon pre-treatment with ROS scavengers, indicating that ROS plays a critical role as an upstream controller in the AMA-induced cell dysfunction.     

In vitro phototoxicity of dihydropyridine derivatives: a photochemical and photobiological study.

Some photosensitizing drugs can cause phototoxic skin responses even after systemic administration; therefore, avoidance of undesired side-effects is a key consideration in drug discovery and development. As a prediction tool for phototoxic risk, we previously proposed the monitoring of reactive oxygen species (ROS) generated from compounds irradiated with UVA/B, which can be effective for understanding photochemical/photobiological properties. In this investigation, we evaluated the photosensitizing properties of a novel dihydropyridine derivative, with bradykinin B(2) receptor antagonist activity (compound A) using our ROS assay and several analytical/biochemical techniques. Exposure of compound A, and several dihydropyridine-type calcium channel antagonists to simulated sunlight resulted in the significant production of singlet oxygen, superoxide, or both, which indicates their photosensitive/phototoxic potential. This is consistent with the observation that compound A under UVA/B light exposure caused significant photodegradation and even peroxidation of fatty acid, which could lead to phototoxic dermatitis. Interestingly, the addition of radical scavengers, especially GSH, MPG and BHA, could attenuate the lipid peroxidation, suggesting the involvement of ROS generation in the phototoxic pathways of compound A. In the 3T3 neutral red uptake phototoxicity test, compound A also showed a phototoxic effect on 3T3 mouse fibroblast cells. These findings also support the usefulness of the ROS assay for the risk assessment studies on the drug-induced phototoxicity even at the early stages of pharmaceutical development.     

Antioxidant strategies for neurodegenerative diseases

A very large body of evidence supports the involvement of oxidative stress in neurodegenerative pathologies. Therefore, therapies based on administration of antioxidant and reactive oxygen species (ROS) scavenging substances have been widely proposed for treatment of both acute and chronic neuropathological diseases. In the present review, several recent patents (mostly from 1998 - 2000) based on the proposed use of antioxidant strategies to treat neurodegenerative diseases are considered. In particular, the following classes of patents are examined: patents for novel antioxidant and ROS scavengers, pharmaceutical compositions in which antioxidant and ROS scavengers are claimed together with other pharmacological tools, patents based on new biotechnological and gene therapy approaches, and patents based on enzymatic manipulation of ROS production and elimination. In the comment, the most promising perspectives stemming from these patents are discussed; in particular: (i.) the usefulness of pharmaceutical compositions able to counteract the deleterious effects of excessive amounts of free radicals originating from different cellular process, without blocking the physiological beneficial action of their controlled production; (ii.) the potential beneficial effects of combining antioxidant treatments with modulation of the activity of receptors for excitatory amino acid and modulation of calcium levels; (iii.) the tremendous future impact of biotechnological and gene therapy techniques.