Library Of Candidates Research Articles

A Study of the Dynamic Interaction of Surfactants with Graphite and Carbon Nanotubes using Fmoc−Amino Acids as a Model System

We have studied the dynamic interaction of surfactants with carbon surfaces by using a series of Fmoc- (N-(fluorenyl-9-methoxycarbonyl)) terminated amino acid derivatives (Fmoc-X, where X is glycine, tyrosine, phenylalanine, tryptophan, or histidine) as a model system. In these systems, highly conjugated fluorenyl groups and aromatic amino acid side chains interact with the carbon surface, while carboxylate groups provide an overall negative charge. Ideal carbon surfaces were selected which possessed either predominantly macroscale (graphite) or nanoscale features (multiwalled carbon nanotube (MWNT) mats). The adsorption equilibrium for the Fmoc-X solutions with the graphitic surfaces was well-described by the Freundlich model. When a library containing various Fmoc-X compounds were exposed to a target graphite surface, Fmoc-tryptophan was found to bind preferentially at the expense of the other components present, leading to a substantial difference in the observed binding behavior compared to individual adsorption experiments. This approach therefore provides a straightforward means to identify good surfactants within a library of many candidates. Finally, the fully reversible nature of Fmoc-X binding was demonstrated by switching the surface chemistry of carbon substrate through sequential exposure to surfactants with increasing binding energies.

Systematic analysis of a simple adaptor protein PDZK1: ligand identification, interaction and functional prediction of complex

PDZK1 is a simple adaptor protein with four protein interaction PDZ domains, but without any other known functional domains. Here, we used yeast two‐hybrid screening of a random peptide library and high‐throughput validation screening of a specialized PDZ ligand candidate library to systematically and comprehensively identify PDZK1 ligands. The potential functional associations of the ligands were predicted by functional annotations from a MILANO literature search and subcellular localizations. The ligands were considered more likely to be functionally associated if they had similar patterns of functions or closely related functions. For some functionally associated ligand pairs, interaction with one ligand was found to be influenced by another ligand in a yeast three‐hybrid system. Many G‐protein signaling pathway‐related proteins were found to interact with PDZK1, and they were likely to be functionally associated with transporters based on their closely related functions. This strategy can be extended to the study of other adaptor proteins that contain peptide‐binding domains.

Combinatorial Evaluation of Cations, pH-sensitive and Hydrophobic Moieties for Polymeric Vector Design

Three combinatorial libraries of polymeric vectors were evaluated to investigate the functional roles of molecular weight (MW), cations, pH-sensitive moieties, and hydrophobic derivitization in polymer-mediated gene delivery. Four cationic and pH-sensitive moieties (imidazole, primary, secondary, and tertiary amino) and three hydrophobic residues (C4 butyl, C6 hexyl, and C8 octyl) were assessed in single and serially incremented, binary combinations. Three MWs were evaluated-10, 30, and 50 kDa. The highest levels of transfection, comparable to branched PEI (25 kDa), were achieved by 30 kDa and 50 kDa formulations containing primary amino and imidazole groups. Primary amino groups offered superior charge-neutralizing and size-condensing capacity, while imidazole groups appeared to bind with DNA via nonelectrostatically mediated interactions to produce stable polyplexes that were resistant to premature dissociation. Eight of the 10 highest-transfecting polymers possessed IC(50) values greater than the maximum concentration of free polymers exposed to cells (200 microg/ml). The results herein have identified highly efficient polymeric formulations with superb toxicity profiles and have revealed the functional roles that the investigated pendant groups play in the transfection process. The reported polymeric system offers a versatile and robust platform upon which future structure-function studies may be based to create safer and more efficient polymeric vectors.

Systematic Analysis of a Simple Adaptor Protein PDZK1: Ligand Identification, Interaction and Functional Prediction of Complex

PDZK1 is a simple adaptor protein with four protein interaction PDZ domains, but without any other known functional domains. Here, we used yeast two-hybrid screening of a random peptide library and high-throughput validation screening of a specialized PDZ ligand candidate library to systematically and comprehensively identify PDZK1 ligands. The potential functional associations of the ligands were predicted by functional annotations from a MILANO literature search and subcellular localizations. The ligands were considered more likely to be functionally associated if they had similar patterns of functions or closely related functions. For some functionally associated ligand pairs, interaction with one ligand was found to be influenced by another ligand in a yeast three-hybrid system. Many G-protein signaling pathway-related proteins were found to interact with PDZK1, and they were likely to be functionally associated with transporters based on their closely related functions. This strategy can be extended to the study of other adaptor proteins that contain peptide-binding domains.

A new decorin‐like tetrapeptide for optimal organization of collagen fibers

Citation: IFSCC Magazine, 10 (2007) (4) 309–314Decorin interacts with collagen via its protein core and influences collagen fibrillogenesis, thus regulating excessive bundle‐like aggregation of collagen. As skin ages, there is a lack of functional decorin which results in disrupted collagen fibers and a reduction in the tensile strength of the skin. Therefore, a substitute for decorin would make up for the non‐functional decorin that is present as we age.Two tetrapeptide sequences were identified as the specific binding sites of decorin to collagen fibrils. These sequences were engineered in order to generate new tetrapeptides with improved affinity that would present a decorin‐like activity. A focused library of several candidates was synthesized containing only tetrapeptides that mimicked the binding sequences of decorin. The candidates were screened with an in vitro collagen fibrillogenesis assay, and the tetrapeptide with the INCI name Tripeptide‐10 Citrulline achieved the best results. Like decorin, this synthetic tetrapeptide proved in in vitro tests to regulate collagen fibrillogenesis and influence the diameter of collagen fibers, making them thinner and more uniform. Tripeptide‐10 citrulline is a new cosmetic active to specifically target collagen fiber organization. Skin collagen quality is addressed rather than skin collagen quantity. Tripeptide‐10 citrulline ensures uniformity of fibril diameter and increases skin suppleness due to a better cohesion of collagen fibers.Keywords: collagen, decorin, diameter, fiber, organizationWinner of the Award for the best Podium Presentation at the IFSCC Conference 2007, Amsterdam, The Netherlands

A new decorin‐like tetrapeptide for optimal organization of collagen fibres

Decorin interacts with collagen via its protein core and influences collagen fibrillogenesis, thus regulating excessive bundle-like aggregation of collagen. As skin ages, there is lack of functional decorin, which results in disrupted collagen fibres and in a reduction in the tensile strength of the skin. Therefore, a substitute for decorin would make up for the non-functional decorin that is present as we age. Two tetrapeptide sequences have been identified as the specific binding sites of decorin to collagen fibrils. These sequences were engineered to generate new tetrapeptides with improved affinity that would present a decorin-like activity. A focused library of several candidates was synthesized containing only tetrapeptides that mimicked the binding sequences of decorin. The candidates were screened with an in vitro collagen fibrillogenesis assay and the tetrapeptide with International Nomenclature of Cosmetic Ingredients (INCI) name Tripeptide-10 Citrulline achieved the best results. Like decorin, this synthetic tetrapeptide proved, through in vitro tests, to regulate collagen fibrillogenesis and to influence the diameter of collagen fibres, making them thinner and more uniform. Tripeptide-10 Citrulline is a new cosmetic active to target specifically collagen fibre organization. Skin collagen quality is addressed rather than skin collagen quantity. Tripeptide-10 Citrulline ensures uniformity in fibril diameter and increases skin suppleness because of a better cohesion of collagen fibres.

Screening human antibody libraries against carcinoma cells by affinity purification and polymerase chain reaction

Bacterial scFv clones from a naïve antibody library have been isolated against cancer cell antigens with AffiSelect, a novel screening method that indirectly identifies candidate library members via an antigen reporter gene. The first step is the coating of carcinoma cell surface epitopes (antigen) with either mAbs, scFvs or phages (library members). Upon binding to a cell surface ligand, the library member generates a linking moiety. This facilitates magnetic affinity purification of the antibody–cancer cell complexes, detected by the polymerase chain reaction (PCR) using the β-actin gene of the cancer cell as the target. Combining these well-known methods resulted in a higher resolution than a comparable cell-based ELISA method of detection. We have isolated human scFv antibodies against surface antigens of a lung carcinoma cell line. These were identified from a polyclonal mixture of phage display-enriched library clones comparing PCR patterns of the carcinoma cell line with the two negative cell types, HUVEC and peripheral blood cells (PBLs). The positive clones were sequenced and verified by FACS.

Screening the Sequence Selectivity of DNA-Binding Molecules Using a Gold Nanoparticle-Based Colorimetric Approach

We have developed a novel competition assay that uses a gold nanoparticle (Au NP)-based, high-throughput colorimetric approach to screen the sequence selectivity of DNA-binding molecules. This assay hinges on the observation that the melting behavior of DNA-functionalized Au NP aggregates is sensitive to the concentration of the DNA-binding molecule in solution. When short, oligomeric hairpin DNA sequences were added to a reaction solution consisting of DNA-functionalized Au NP aggregates and DNA-binding molecules, these molecules may either bind to the Au NP aggregate interconnects or the hairpin stems based on their relative affinity for each. This relative affinity can be measured as a change in the melting temperature (Tm) of the DNA-modified Au NP aggregates in solution. As a proof of concept, we evaluated the selectivity of 4',6-diamidino-2-phenylindone (an AT-specific binder), ethidium bromide (a nonspecific binder), and chromomycin A (a GC-specific binder) for six sequences of hairpin DNA having different numbers of AT pairs in a five-base pair variable stem region. Our assay accurately and easily confirmed the known trends in selectivity for the DNA binders in question without the use of complicated instrumentation. This novel assay will be useful in assessing large libraries of potential drug candidates that work by binding DNA to form a drug/DNA complex.

Domino syntheses of bioactive tetronic and tetramic acids

Natural products containing tetronic acid or tetramic acid moieties continue to attract the interest of chemists, biologists, and physicians due to their challenging structures and to the wide range of biological activities they display. This review portrays the structural varieties of tetronic and tetramic acids and the spectrum of possible therapeutically relevant effects in man for exemplary derivatives. Their biosynthetic origin from alpha-amino and alpha-hydroxy acids is briefly discussed as is the relationship between their structures and their modes of interaction with biochemical effectors such as metal cations or enzymes. A short overview of laboratory syntheses of the heterocyclic core structures of tetramic and tetronic acids is provided with an emphasis on those emulating the biosynthesis. A synthesis from the alpha-amino or alpha-hydroxy esters and the cumulated phosphorus ylide Ph(3)PCCO based upon a domino addition-intra-Wittig alkenation sequence is presented with applications to the preparation of the antibiotics reutericyclin and tenuazonic acid, the cytotoxic melophlin B, and the enzyme inhibitor RK-682. Procedural advantages of immobilizing either starting component by attaching it to a resin and its exploitation in the parallel synthesis of libraries of potential drug candidates are described. The basic domino reaction can even be extended by further C-C bond forming steps when starting from suitable alpha-hydroxy or alpha-amino allyl esters. Depending on the chosen reaction conditions, bioactive intermediates of formally three to seven step long cascades can be obtained. Among them, herbicidal 3-alkyltetronic acids and lactone endoperoxides with antiplasmodial activity exceeding that of the natural antimalarial lead artemisinin. Hence, this domino reaction gives access to diversely functionalized derivatives of tetronic and tetramic acids. As it can also be ported to solid phase, it is ideally suited for parallel and combinatorial processing. Future developments might include running such domino sequences in continuous mode in arrays of "labs on microchips".

A High-Throughput Screen for Compounds That Inhibit Aggregation of the Alzheimer’s Peptide

Aggregation of the Alzheimer's peptide Abeta produces toxic multimeric species that play a key role in the development of Alzheimer's disease. Compounds that inhibit this aggregation may prove useful as therapeutic agents for the prevention or treatment of Alzheimer's disease. Although aggregation inhibitors may already exist in combinatorial libraries, finding these compounds in a cost-effective high-throughput manner poses an enormous challenge. To meet this challenge, we have developed a novel high-throughput screen capable of isolating inhibitors of Abeta aggregation from large libraries of inactive candidates. The screen uses a fusion of Abeta42 to GFP. In the absence of inhibition, the rapid misfolding and aggregation of Abeta42 causes the entire fusion protein to misfold, thereby preventing fluorescence. Compounds that inhibit Abeta42 aggregation enable GFP to fold into its native structure and be identified by the resulting fluorescent signal. By implementing the screen on a pilot library of triazine derivatives, we have identified several putative inhibitors. One of the selected compounds was studied in detail by a series of biochemical and biophysical methods. These studies confirmed that the selected compound inhibits aggregation of synthetic Abeta42 peptide. The fluorescence-based method described here is rapid and inexpensive and can be used to screen large libraries for inhibitors of Abeta42 aggregation and/or amyloidogenesis.

A Microcapillary System for Simultaneous, Parallel Microwave‐Assisted Synthesis

A continuous flow, microwave-assisted, parallel-capillary microreactor has been developed. Libraries of drug candidates were prepared on the milligram scale with this reactor by injecting plugs of reagents from separate syringes into common reaction capillaries, thereby producing discrete compounds in excellent yield and purity. Microwave irradiation provides the necessary energy that existing room-temperature microreactor technology lacks for higher activation barrier transformations, producing the required amounts of desired compounds in minutes or less.

Polyplexes and lipoplexes for mammalian gene delivery: from traditional to microarray screening.

Gene therapy requires the development of non-toxic and highly efficient delivery systems for DNA and RNAi. Polycations, especially dendrimers, have shown enormous potential as gene transfer vehicles, displaying minimal toxicity with a broad range of cell lines. In this paper, a total of 13 dendrimers, up to G3.0, were constructed from AB(3) type isocyanate monomers using solid phase methodology and evaluated for transfection activity. Among the library of compounds prepared, a G3.0 dendrimer displayed comparable activity to Superfect. Gel retardation assays demonstrated that all of the compounds completely bound plasmid DNA, indicating the efficient formation of complexes between DNA and the dendrimers. A "transfection microarray" approach was developed for screening these compounds as well as a panel of lipoplexes (complexes of DNA with cationic lipids) and polyplexes (complexes of DNA with synthetic polycationic polymers), in 3D solution like micro-assay). Five cationic lipids with a cholesterol tail showed stronger or comparable transfection activity relative to Effectene. The new, micro-array screening method was rapid and miniaturized, offering the potential of high throughput screening of large libraries of transfection candidates, with thousands of library members per array, and the ability to rapidly screen a broad range of cell types.

Generation of new protein kinase inhibitors utilizing cytochrome p450 mutant enzymes for indigoid synthesis.

Indigoids, a class of bis-indoles, represent a promising protein kinase inhibitor scaffold. Oxidation of indole by cytochrome P450 (P450) has been shown to generate species (indoxyl, isatin) that couple to yield indigo and indirubin. Escherichia coli-expressed human P450 2A6 mutants isolated from a randomized library were incubated with 27 substituted indole derivatives. Extracts of the cultures were screened for inhibition of human cyclin-dependent kinases (CDK)-1 and -5 and glycogen synthase kinase-3 (GSK3). The extracts from cultures incubated with 5-methoxyindole were the most inhibitory. High-performance liquid chromatography (HPLC) separation yielded a mixture of seven colored indigoids. These indigoids included indigo, indirubin, the di(5-methoxy) derivatives of indigo and indirubin, and both of the possible mono 5-methoxy derivatives of indirubin, which were all identified by visible, mass, and NMR spectra. Cultures with 5-methylindole added to the media also yielded inhibitory material, and 5- and 5'-methylindirubin were characterized. The most inhibitory of these indigoids were the monosubstituted indirubins and 5,5'-dimethoxyindirubin, which was > or =10x more active than indirubin. Thus, the overall approach involves the use of a library of randomized enzyme mutants to activate component moieties of a desired set of larger molecules, thus yielding a library of drug candidates that can be screened and characterized. The general strategy may have additional applications.

Design of diverse and focused combinatorial libraries using an alternating algorithm.

There is considerable research in chemistry to develop reaction conditions so that any of a very large number of reactants will successfully form new compounds, e.g. for two components, A(i) + B(j) --> A-B(ij). The numbers of A's and B's usually make it impossible to make all the possible products; with multicomponent reactions, there could easily be millions to billions of possible products. There is a need to identify subsets of reagents so that the resulting products have desirable predicted properties. Our idea is to select reactants sequentially and iteratively to optimize the evolving candidate library. The new Alternating Algorithm, AA, can be used for diversity, a space-filling design, or for a focused design, using either a near neighborhood or structure-activity relationship, SAR. A diversity design seeks to select compounds different from one another; a focused design seeks to find compounds similar to an active compound or compounds that follow a structure activity relationship. The benefit of the method is rapid computation of diversity or focused combinatorial chemical libraries.

Dynamic high throughput screening of chemical libraries using acoustic-wave sensor system

We report a novel sensor-based high throughput screening (HTS) system for identification and quantitation of volatile substances in combinatorial chemical libraries. The measurement method employs a combination of a periodic introduction of a minute amount of a liquid sample into the HTS system, rapid evaporation of volatile components in the sample at room temperature, and dynamic measurement of a generated vapor pulse. These measurements are performed using an array of four 10 MHz acoustic-wave thickness-shear mode sensors coated with different chemically sensitive films. Developed HTS system is applied for screening of multiple samples such as those created in combinatorial chemical libraries of catalyst candidates in an industrially important arene oxidation process. The temporal modulation of the concentration of analyte vapors and measurement of both the temporal profile and the magnitude of the response improves sensor selectivity and makes possible robust identification and quantitation of arene oxidation components such as cresol and benzoquinone in multicomponent combinatorial mixtures with reduced number of sensors in the array. Different solvents such as water, acetonitrile, benzene, and toluene do not alter the response of sensors to analytes. Depending on the gas flow rate, quantitative measurements are performed 10–150 s after the sample introduction and provide significant throughput advantage over gas-chromatographic instruments. Determinations of mixtures of analytes in a variety of solvents are performed using multivariate locally weighted regression. This data analysis method provides the root mean squared error of prediction of less than 2 μg when measurements of cresol and benzoquinone amounts ranging from 0 to 50 μg are performed in 2 μL samples. This method of dynamic sensor-based measurements allows for instrument miniaturization and increases the usefulness of the instrument in space-limited applications. Upon operation of multiple sensors in parallel, effective analysis of dense combinatorial libraries of materials is possible.

Approaches to library design for combinatorial chemistry.

With the advent of high-throughput technologies in drug discovery, combinatorial chemistry has superseded natural products as the prime source of compounds to be tested in biological screening (). The reason being that compared with classical synthesis, high-throughput chemistry can produce a much larger number of compounds within a much shorter period of time. However, owing to the combinatorial nature of the problem and the large number of reagents available, many more compounds are theoretically accessible via a given synthetic route than it is actually feasible to synthesize. Therefore, library design techniques are employed to identify those reagents that yield a library enriched with the desired properties. Early on, most applications of these techniques concentrated on designing very diverse libraries, with the idea that these libraries would be tested against a variety of biological assays. The ideal library in this case would be with no voids, no redundancy, and an even distribution with regard to a given chemical space. Nowadays, it is equally common to design targeted libraries to have a maximal activity in a selected assay. Here, all compounds would be designed to be similar to given hits or leads. Other applications of library design techniques include the computational evaluation of templates, i.e., prioritization of candidate libraries for synthesis and screening (see Note 1).

Techview: drug discovery. Integrating combinatorial synthesis and bioassays.

The increasing demand for large chemical libraries of potential drug candidates is accompanied by the need for efficient methods for identifying biologically active compounds in these libraries. In this TechView, Rademann and Jung (p. 1947) discuss strategies for creating efficient interfaces between combinatorial synthesis and screening and hereby extracting biologically relevant information from libraries of chemical compounds.

Applications of random sampling to virtual screening of combinatorial libraries

We describe statistical techniques for effective evaluation of large virtual combinatorial libraries (>1010 potential compounds). The methods described are used for computationally evaluating templates (prioritization of candidate libraries for synthesis and screening) and for the design of individual combinatorial libraries (e.g., for a given diversity site, reagents can be selected based on the estimated frequency with which they appear in products that pass a computational filter). These statistical methods are powerful because they provide a simple way to estimate the properties of the overall library without explicitly enumerating all of the possible products. In addition, they are fast and simple, and the amount of sampling required to achieve a desired precision is calculable. In this article, we discuss the computational methods that allow random product selection from a combinatorial library and the statistics involved in estimating errors from quantities obtained from such samples. We then describe three examples: (1) an estimate of average molecular weight for the several billion possible products in a four-component Ugi reaction, a quantity that can be calculated exactly for comparison; (2) the prioritization of four templates for combinatorial synthesis using a computational filter based on four-point pharmacophores; and (3) selection of reagents for the four-component Ugi reaction based on their frequency of occurrence in products that pass a pharmacophore filter.

Studies towards the identification of potent, selective and bioavailable thrombin inhibitors

The application of selection criteria, based on potency and physicochemical parameters, to a candidate library of thrombin inhibitors is described. The utility of the approach is exemplified by the discovery of a potent, selective and bioavailable thrombin inhibitor 62.

Isocyanide Based Multi Component Reactions in Combinatorial Chemistry

Although usually regarded as a recent development, the combinatorial approach to the synthesis of libraries of new drug candidates was first described as early as 1961 using the isocyanide-based one-pot multicomponent Ugi reaction. Isocyanide-based multi component reactions (MCR's) markedly differ from their usual two component counterparts. In the context of combinatorial chemistry, they offer more structural variations, more accessible compounds and more diversity than any other previously known reaction. This review examines the history of isocyanide based MCR's and their applicability in combinatorial chemistry.