Combinatorial Chemistry & High Throughput Screening - Volume 9, Issue 6, 2006

Free radicals are atoms or groups of atoms with an odd (unpaired) number of electrons and can be formed when oxygen interacts with certain molecules. Once formed these highly reactive radicals can start a chain reaction. Their danger comes from the damage they can do when they react with important cellular components such as DNA, or the cell membrane. Increasingly, their role in a number of human disease processes is being accepted, although conclusive evidence is lacking in many cases, due to the difficulties in detecting free radicals that have very short lifetimes. To prevent free radical damage the body has a defense system of antioxidants. Antioxidants interact with and stabilize free radicals and can prevent some of the damage free radicals otherwise might cause. Antioxidants are intimately involved in the prevention of cellular damage, cancer, aging, and a variety of diseases. However, these benefits have not been confirmed by recent large clinical trials. Research suggests that consumption of antioxidant-rich foods reduces damage to cells and biochemicals from free radicals. This can slow down, prevent, and even reverse certain diseases that result from cellular damage, and perhaps even slow down the natural aging process. An antioxidant may act by catalysis, or, in cells, it may act as a cofactor that transfers reducing equivalents from NADPH. Finding of novel drugs is a very difficult process. Combinatorial chemistry is one of the important methodologies that can help to reduce the time and costs associated with producing new drugs. In the process of finding new drug candidates medicinal chemists nowadays have a variety of options from which to choose, and one is to apply combinatorial chemistry techniques. It is now possible to produce compound libraries to screen for novel bioactivities. In the preparation of drug candidates, the automated, and combinatorial use of chemical building blocks facilitates the generation and screening of large numbers of compounds. Combinatorial chemistry has been proven to be an efficient way of generating libraries of compounds and can be used to identify lead compounds in a short period of time. It is relatively unexplored when considering the discovery of novel anti-oxidative agents. The purpose of this special issue is to serve as a guide to what antioxidants are and to briefly review their role in general health. Recent developments of melatonin related antioxidant compounds, antioxidant containing foods especially in the Black Sea region of Turkey, evaluation and comparison of the ROS generating and LP inducing effect of several halogenated aromatic compounds, the relationship between antioxidants and inflammatory diseases, and finally, combinatorial library synthesis of antioxidant compounds are addressed in this special issue. I would like to thank the authors for their contributions and the editor of Combinatorial Chemistry & High Throughput Screening for the invitation to act as guest editor for this special issue.

Melatonin is known for its radical scavenger activity, which is related to its ability to protect cells from different kinds of oxidative stress. Oxidative stress has been implicated in the development of neurodegenerative diseases like Parkinson, Alzheimer's disease, Huntington's disease, epileptic seizures, stroke, and as a contributor to aging and some cancer types. The antioxidant properties of melatonin include scavenging free radicals and the regulation of the activity and expression of antioxidant and pro-oxidant enzymes. Due to its free radical scavenger and antioxidant properties, multiple melatonin-related compounds such as melatonin metabolites and synthetic analogues are under investigation to determine which exhibit the highest activity with the lowest side effects. This review addresses recent studies with melatonin and related compounds.

Since oxidative cellular damage contributes to the development of cancers, heart disease and ageing, the synthesis of antioxidative agents which are able to either prevent or mitigate oxidative stress to cells is an important area of investigation. Combinatorial chemistry has had a profound impact on the discovery and optimisation of potential lead compounds, especially in the medicinal field. This review details recent examples of combinatorial chemistry dealing with the synthesis of novel antioxidants with an emphasis on solid phase compound synthesis and parallel library synthesis.

Oxidants play a significant role in the pathogenesis of a number of disorders such as inflammation, rheumatoid arthritis, asthma, psoriasis and contact dermatitis leading to oxidative stress. Oxidative stress may be defined as an imbalance between cellular production of reactive oxygen species (ROS) and antioxidant defense mechanisms. ROS (e.g., superoxide radical, peroxynitryl, hydroxyl radical and hydrogen peroxide) are constantly produced as a result of metabolic reactions in living systems. The aim of this review is to describe recent developments in the study of antioxidants and their role in preventing the formation of ROS. The processes associated with inflammatory responses are complex and often involve ROS. There are many mediators, which initiate and amplify the inflammatory response such as histamine, serotonin, pro-inflammatory cytokines (interleukin-1B (IL-1b) and tumor necrosis factor (TNF-α)), inflammatory cells (leukotrienes, macrophages), metabolic products of arachidonic acid (thomboxane A2, prostaglandins and leukotrienes). The first part of this review focuses on the role of ROS in inflammation. The second part concerns synthetic antioxidants with antiinflammatory activity, and the third part addresses naturally occurring antioxidants with antiinflammatory activity.

The ethanol extracts of Morchella vulgaris (EEMV) and Morchella esculanta (EEME) were analysed for their antioxidant activities in different systems including reducing power, free radical scavenging, superoxide anion radical scavenging, total antioxidant activity, and metal chelating activity. EEMV and EEME had similar reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, and metal chelating activity at concentrations of 50, 100, and 150 μg/mL. These various antioxidant activities were compared to standard antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and α-tocopherol. The percent inhibition of different concentrations of EEMV on peroxidation in the linoleic acid system was 85 and 87 % respectively, which was greater than that of 100 and 250 μg/mL of α-tocopherol (50 and 77%, respectively) and similar to 250 μg/mL of BHA (85, 87%, respectively). The percent inhibition of different concentrations of EEME on peroxidation in the linoleic acid system was 80 and 87 % respectively, which was greater than that of 100 and 250 μg/mL of α-tocopherol (50, 77%) and similar to 250 μg/mL BHA (87%). On the other hand, the percent inhibition of 100 and 250 μg/mL of BHT was 97 and 99%, respectively. In addition, the total phenolic compounds in EEMV and EEME were determined as gallic acid equivalents.

The present study was designed to investigate the potential of reactive oxygen species (ROS) generating and subsequent ROS-mediated lipid peroxidation (LPO) inducing effect of several mono- and di-halogenated biphenyls and biphenyl ethers in rat hepatocytes in vitro. For this aim, 4-chloro- and 4-bromo biphenyl (4-CB and 4-BB), 4-OH, 4'-BB, 4-bromo diphenylether (4-BDE), 4,4'-dichlorobiphenyl (4,4'-DCB), 4,4'-dibromobiphenyl (4,4'-DBB), and 3,4- dichlorobiphenyl (3,4-DCB) were incubated with freshly isolated rat hepatocytes. Their oxidative potential was evaluated by detecting the intracellular ROS formation by oxidant-sensing fluorescent probes (2',7'-dichlorofluorescein diacetate and C11-BODIPY 581/591 ) using a multiplate reader and determining the levels of eight LPO products (formaldehyde, malondialdehyde, propanal, butanal, pentanal, hexanal, octanal, and nonanal) by a gas chromatography-electron capture detection. 4-BDE was found to be active both in cytoplasm and in the cell membrane in terms of inducing the formation of ROS. Another important finding was the increase in ROS-inducing potential of 4-BB when the same concentration of the hydroxylated derivative, 4-OH,4'-BB, was incubated with hepatocytes. 4-BDE was also found to be the most effective among all tested compounds in inducing LPO where 4-OH, 4'-BB was again more potent than its unmetabolized form, 4- BB. Lactate dehydrogenase leakage analyses indicated that all tested compounds are cytotoxic; 4-BDE caused the highest LDH leakage compared to other mono-halogenated biphenyls tested. Our results suggest that ROS formation by chlorinated biphenyls and mono-hydroxylated bromobiphenyls, and concomitant induction of LPO might be involved in the cytotoxic effects of these industrial pollutants. Similar effects of mono-BDE are also reported, which is a novel observation.

The objective of this work was to apply artificial neural networks (ANNs) to the classification group of 43 derivatives of phenylcarbamic acid. To find the appropriate clusters Kohonen topological maps were employed. As input data, thermal parameters obtained during DSC and TG analysis were used. Input feature selection (IFS) algorithms were used in order to give an estimate of the relative importance of various input variables. Additionally, sensitivity analysis was carried out to eliminate less important thermal variables. As a result, one classification model was obtained, which can assign our compounds to an appropriate class. Because the classes contain groups of molecules structurally related, it is possible to predict the structure of the compounds (for example the position of the substitution alkoxy group in the phenyl ring) on the basis of obtained parameters.

The goal of combinatorial chemistry is to simultaneously synthesize sets of compounds possessing properties that are then distinguished through screening. As the size of a compound set increases, data analysis becomes more challenging. Analysis of Variance (ANOVA) is an accepted statistical method that offers a straightforward solution to this problem. Two steps encountered by combinatorial scientists appear well suited to ANOVA: the prediction of synthetic outcomes (purity and yield) of set members and the analysis of screening data to identify combinations of reagent inputs that result in molecules with a desired property. To illustrate, a subset of a combinatorial array, referred to as a reaction rehearsal set, is evaluated to create a model predictive of the individual synthetic outcomes of the full matrix. In a second exercise, the biochemical screening data obtained from a combinatorial library is analyzed to identify reagent interactions that result in molecules possessing the sought activity.

In the computer-aided drug design, in order to find some new leads from a large library of compounds, the pattern recognition study of the diversity and similarity assessment of the chemical compounds is required; meanwhile in the combinatorial library design, more attention is given to design target focusing library along with diversity and drug-likeness criteria. This review presents the current state-of-art applications of Kohonen self-organizing maps (SOM) for studying the compounds pattern recognition, comparing the property of molecular surfaces, distinguishing drug-like and nondrug- like molecules, splitting a dataset into the proper training and test sets before constructing a QSAR (Quantitative Structural-Activity Relationship) model, and also for the combinatorial libraries comparison and the combinatorial library design. The Kohonen self-organizing map will continue to play an important role in drug discovery and library design.

We have developed a high throughput assay for the measurement of protease activity in solution. This technology will accelerate research in functional proteomics and enable biologists to streamline protease substrate evaluation and optimization. The peptide sequences that serve as protease substrates in this assay are labeled on the carboxy terminus with a biotin moiety and a fluorescent tag is attached to the amino terminus. Protease cleavage causes the biotin containing fragment to be detached from the labeled peptide fragment. Following the protease treatment, all biotin containing species (uncleaved substrates and the cleaved carboxy terminal fragment of the substrate) are removed by incubation with streptavidin beads. The cleaved fluorescently labeled amino terminal part of the substrate remains in solution. The measured fluorescence intensity of the solution is directly proportional to the activity of the protease. This assay was validated using trypsin, chymotrypsin, caspase-3, subtilisin-A, enterokinase and tobacco etch virus protease.

Dr. Sibel Suzen studied at Ankara University, from where she received her Pharmacy degree (1985) and Masters degree in Pharmaceutical Chemistry (1989). She obtained a Ph. D. (1997) from University of Swansea, Chemistry Department (UK) under the supervision of Dr. J. M. Williams where she worked on the synthesis and hydrazinolysis of dehydroalanine derivatives in order to investigate possible relevance to glycoprotein hydrazinolysis. She has been involved in several medicinal chemistry projects related aldose reductase inhibitory compounds, antioxidant compounds, biologically active indole derivatives, radioprotective agents, and electroanalytical investigation of in vitro drug metabolism. She has organized and participated in organizing committees of numerous international meetings related to pharmaceutical and medicinal chemistry. Her research interest focuses on the synthesis and biological evaluation of melatonin and peptide related antioxidant compounds as well as aldose reductase inhibitors and radioprotective agents. She is currently Associate Professor of Pharmaceutical Chemistry of the Faculty of Pharmacy, Ankara University.