Mini-review: Combinatorial approaches for the design of novel coating systems

Abstract

Combinatorial and high throughput experimental methods are being applied to the design and development of novel polymers and coatings used in a number of application areas. Methods have been developed for polymer synthesis and screening and for the development of polymer thin film and coating libraries and the screening of these libraries for key properties such as surface energy and modulus. Combinatorial and high throughput methods enable the efficient exploration of a large number of compositional variables over a wide range. In the development of coatings for use in the marine environment, the key challenge is in the development of screening methods that can predict good performance. A number of assays are under development that will permit the rapid screening of the interaction of coatings with representative marine organisms.     

Combinatorial chemistry in the agrosciences

Combinatorial chemistry and high throughput screening have had a profound effect upon the way in which agrochemical companies conduct their lead discovery research. The article reviews recent applications of combinatorial synthesis in the lead discovery process for new fungicides, herbicides and insecticides. The role and importance of bioavailability guidelines, natural products, privileged structures, virtual screening and X-ray crystallographic protein structures on the design of solid- and solution-phase compound libraries is discussed and illustrated.     

Toxicity Screening of a Combinatorial Library: Correlation of Cytotoxicity and Gene Induction to Compound Structure

Combinatorial chemistry has increased the number of compounds available for efficacy and safety assessment by several orders of magnitude and has made high throughput assays essential. To test whether higher throughput toxicity assays could be of utility in screening compounds in early development, a selected set of combinatorial chemistry compounds was screened for induction of 70-Kd heat shock protein (HSP70) and 45-Kd growth arrest and DNA damage protein (GADD45) mRNA levels as well as cytotoxicity, in HepG2 cells, using a 96-well microtiter plate format. Both assays, the branched DNA (Quantigene) assay for mRNA levels and MTT for cytotoxicity, were robust enough to be incorporated into a screening format using a single replicate and a single concentration of compound. Significantly, a structure/toxicity correlation was established with this set of compounds with cytotoxicity and gene induction patterns linked to compound structure. Therefore, this type of early screening may be useful in identifying toxic substituents, enabling the design of libraries with less potential for toxicity. While structure/toxicity correlations were observed, no relationship was observed between GADD45 gene induction and mutagenesis as measured by the Ames bacterial reverse mutation assay.     

随机突变文库构建与筛选研究进展

定向进化是一个循环过程,在构建多样化基因序列和筛选功能基因变体之间交替进行.该技术目前已被广泛应用于DNA序列、基因功能和蛋白质结构的优化和分析.定向进化包括随机基因文库的生成、基因在合适宿主中的表达和突变文库的筛选.构建基因文库的关键是库容量和突变多样性,而筛选变体的关键是高灵敏度和高通量.文中讨论了定向进化技术的最...     

Microfluidic synthesis of composite cross‐gradient materials for investigating cell–biomaterial interactions

Combinatorial material synthesis is a powerful approach for creating composite material libraries for the high‐throughput screening of cell–material interactions. Although current combinatorial screening platforms have been tremendously successful in identifying target (termed “hit”) materials from composite material libraries, new material synthesis approaches are needed to further optimize the concentrations and blending ratios of the component materials. Here we employed a microfluidic platform to rapidly synthesize composite materials containing cross‐gradients of gelatin and chitosan for investigating cell–biomaterial interactions. The microfluidic synthesis of the cross‐gradient was optimized experimentally and theoretically to produce quantitatively controllable variations in the concentrations and blending ratios of the two components. The anisotropic chemical compositions of the gelatin/chitosan cross‐gradients were characterized by Fourier transform infrared spectrometry and X‐ray photoelectron spectrometry. The three‐dimensional (3D) porous gelatin/chitosan cross‐gradient materials were shown to regulate the cellular morphology and proliferation of smooth muscle cells (SMCs) in a gradient‐dependent manner. We envision that our microfluidic cross‐gradient platform may accelerate the material development processes involved in a wide range of biomedical applications. Biotechnol. Bioeng. 2011; 108:175–185. © 2010 Wiley Periodicals, Inc.     

Large-scale growth of the Plasmodium falciparum malaria parasite in a wave bioreactor

We describe methods for the large-scale in vitro culturing of synchronous and asynchronous blood-stage Plasmodium falciparum parasites in sterile disposable plastic bioreactors controlled by wave-induced motion (wave bioreactor). These cultures perform better than static flask cultures in terms of preserving parasite cell cycle synchronicity and reducing the number of multiple-infected erythrocytes. The straight-forward methods described here will facilitate the large scale production of malaria parasites for antigen and organelle isolation and characterisation, for the high throughput screening of compound libraries with whole cells or extracts, and the development of live- or whole-cell malaria vaccines under good manufacturing practice compliant standards.     

Micro-colony array based high throughput platform for enzyme library screening

Enzymes are becoming increasingly important tools for synthesizing and modifying fine and bulk chemicals. The availability of biocatalysts which fulfil the requirements of industrial processes is often limited. Recruiting suited enzymes from natural (e.g. metagenomes) and artificial (e.g. directed evolution) biodiversity is based on screening libraries of microbial clones expressing enzyme variants. However, exploring the complex diversity of such libraries needs efficient screening methods. Overcoming the "screening bottleneck" requires rapid high throughput technology allowing the analysis of a large diversity of different enzymes and applying different screening conditions. Facing these facts an efficient and cost effective method for high throughput screening of large enzyme libraries at the colony level was developed. Therefore, ordered high density micro-colony arrays were combined with optical sensor technology and automated image analysis. The system generally allows the simultaneous monitoring of enzyme activities reflected by up to 7000 micro-colonies spotted on a filter in the size of a micro-titer plate. A developed replica option also allows the analysis of clones under varying external conditions. The method was verified by a model screening using esterases and was proved to provide reliable enzyme activity measurements within single micro-colonies allowing the discrimination of activity differences in the range of 10-20%.     

Combinatorial synthesis of natural products

Combinatorial syntheses allow production of compound libraries in an expeditious and organized manner immediately applicable for high-throughput screening. Natural products possess a pedigree to justify quality and appreciation in drug discovery and development. Currently, we are seeing a rapid increase in application of natural products in combinatorial chemistry and vice versa. The therapeutic areas of infectious disease and oncology still dominate but many new areas are emerging. Several complex natural products have now been synthesised by solid-phase methods and have created the foundation for preparation of combinatorial libraries. In other examples, natural products or intermediates have served as building blocks or scaffolds in the synthesis of complex natural products, bioactive analogues or designed hybrid molecules. Finally, structural motifs from the biologically active parent molecule have been identified and have served for design of natural product mimicry, which facilitates the creation of combinatorial libraries.     

One-bead–one-structure combinatorial libraries

Combinatorial libraries employing the one-bead–one-compound technique are reviewed. Two distinguishing features characterize this technique. First, each compound is identified with a unique solid support, enabling facile segregation of active compounds. Second, the identity of a compound on a positively reacting bead is elucidated only after its biological relevance is established. Direct methods of structure identification (Edman degradation and mass spectroscopy) as well as indirect “coding” methods facilitating the synthesis and screening of nonpeptide libraries are discussed. Nonpeptide and “scaffold” libraries, together with a new approach for the discovery of a pentide binding motif using a “library of libraries,” are also discussed. In addition, the ability to use combinatorial libraries to optimize initially discovered leads is illustrated with examples using peptide libraries. © 1994 John Wiley & Sons, Inc.     

Going native: Direct high throughput screening of secreted full-length IgG antibodies against cell membrane proteins

Gel microdroplet – fluorescence activated cell sorting (GMD-FACS) is an innovative high throughput screening platform for recombinant protein libraries, and we show here that GMD-FACS can overcome many of the limitations associated with conventional screening methods for antibody libraries. For example, phage and cell surface display benefit from exceptionally high throughput, but generally require high quality, soluble antigen target and necessitate the use of anchored antibody fragments. In contrast, the GMD-FACS assay can screen for soluble, secreted, full-length IgGs at rates of several thousand clones per second, and the technique enables direct screening against membrane protein targets in their native cellular context. In proof-of-concept experiments, rare anti-EGFR antibody clones were efficiently enriched from a 10,000-fold excess of anti-CCR5 clones in just three days. Looking forward, GMD-FACS has the potential to contribute to antibody discovery and engineering for difficult targets, such as ion channels and G protein-coupled receptors.     

组合生物催化

自然界最有效的分子是由酶催化的反应所产生,并对这些产物进行自然选择,使其具有优化的生理活性,组合生物催化（Combinatorial Biocatalysis)利用酶反应的多样性,完成有机库（Organic Library)的反复合成,这些反复的反应,可以用分离的酶或全细胞,在天然或非天然的环境中、在溶液或固相中与底物进行反应。组合生物催化是组合方法的在药物发现和发展中产生和优化先导化合物（LeadCompound)的一个有力补充。     

Liquid phase parallel synthesis of guanidines.

Combinatorial synthesis of N,N'-di(Boc)-Protected guanidines containing piperazine and pyrrolidine scaffolds has been developed. We initiate a preliminary study on the reactivity of several guanylating reagents with soluble polymer-bound diamines in liquid phase. Guanidines are liberated from the polymer support under mild conditions in high yields and high purity by simple precipitation and washings. This combinatorial liquid-phase methodology proves to be a useful tool for constructing guanidine libraries containing diamine scaffolds.     

Solution-phase library generation: methods and applications in drug discovery

Solution-phase high throughput synthesis has emerged as a powerful method for the rapid generation of chemical libraries. The success of this approach is largely due to the development of novel synthetic methodologies that expedite the preparation of compounds. Several isolation/purification techniques have also been developed to eliminate the time-consuming purification procedures often associated with solution-phase chemistry. These methods are amenable to parallel synthesis and combinatorial strategies and can be fully automated. In addition, the compound libraries generated using solution-phase high throughput synthesis have been used to accelerate both lead identification and lead optimization programs at various companies.     

Towards proteome-wide production of monoclonal antibody by phage display

Sequencing of the human genome reveals that there are approximately 30,000 genes that encode an even greater number of proteins which comprise the human proteome. Characterization of gene products at the genome-wide scale requires the development of high throughput methods to generate temporo-spatial information on each and every protein in the cell under normal and pathological conditions. Monoclonal antibodies are important reagents for these studies. We have developed a method to generate human monoclonal antibodies by selecting phage antibody libraries directly on antigen blotted onto poly(vinylidene fluoride) membranes. Cellular proteins are first separated by two dimensional (2D) gel electrophoresis, Western blotted onto poly(vinylidene fluoride) membranes, and used to select phage antibody libraries. Monoclonal antibodies can be generated against individual protein spots on a 2D gel. The antibodies are functional in Western blotting, ELISA, and immunohistochemistry. Automation of this process should allow high throughput production of monoclonal phage antibodies against cellular proteins as well as proteins that are uniquely expressed under pathological conditions.     

High throughput magnetic resonance imaging for evaluating targeted nanoparticle probes.

The ability to image specific molecular targets in vivo would have significant impact in allowing earlier disease detection and in tailoring molecular therapies. One of the rate-limiting steps in the development of novel compounds as reporter probes has been the lack of cell-based, biologically relevant, high throughput screening methods. Here we describe the development and validation of magnetic resonance imaging (MRI) as a technique to rapidly screen compounds that are potential MR reporter agents for their interaction with specific cellular targets. We show that MR imaging can (1) evaluate thousands of samples simultaneously and rapidly, (2) provide exceedingly accurate measurements, and (3) provide receptor binding/internalization data as validated by radioactive assays. The technique allows the screening of libraries of peptide-nanoparticle conjugates against target cells and the identification of conjugates that may be subsequently used as reporter agents in vivo. The technology should greatly accelerate the development of target-specific or cell-specific MR contrast agents.     

Minireview: Challenges of High Throughput Screening Against Cell Surface Receptors

Abstract

Excessive or inappropriate activation of cell surface receptors can mediate the development of disease. Receptors, therefore, are a focus for drug discovery activities. Empirical screening is important in the search for novel compounds acting at receptors. Technical developments and the application of molecular biology have facilitated access to receptors of interest and have provided efficient screening methods capable of very high throughput. Reliability in high throughput screening requires the use of appropriate methodology, good screen design and effective validation and quality control processes. Validation should aim to establish that the basic experimental design is sound. In developing software to handle high throughput screening data, a fundamental requirement is to provide performance monitoring and error trapping facilities. Additional requirements are automatic data capture from instruments, on-line data reduction and analysis and transfer of results to central databases. As data volumes increase through effective high throughput screening, conventional interrogation methods become less appropriate and are being augmented by newer computing techniques referred to as knowledge mapping or database mining. Targeting cell surface receptors has been very successful as an approach to drug discovery. If the challenges of high throughput empirical screening are addressed effectively, cell surface receptors will provide new opportunities for improved therapy in the coming years.     

Marcel Faber Roundtable: Is our antibiotic pipeline unproductive because of starvation, constipation or lack of inspiration?

There are few new antibiotics in the pipeline today. The reasons may include starvation at the front of the pipeline due to inadequate sources of suitable compounds to screen coupled with poorly validated discovery methodologies. A successful antibiotic discovery approach in the past, based upon whole cell antibiotic screening of natural products from actinomycetes and fungi, eventually suffered from constipation in the middle of the pipeline due to rediscovery of known compounds, even though low throughput methodology was employed at the front end. The current lack of productivity may be attributed to the poor choice of strategies to address the discovery of new antibiotics. Recent applications of high throughput in vitro screening of individual antibacterial targets to identify lead compounds from combinatorial chemical libraries, traditional chemical libraries, and partially purified natural product extracts has not produced any significant clinical candidates. The solution to the current dilemma may be to return to natural product whole cell screening. For this approach to work in the current millennium, the process needs to be miniaturized to increase the throughput by orders of magnitude over traditional screening, and the rediscovery of known antibiotics needs to be minimized by methods that can be readily monitored and improved over time.     

Physico-chemical and biological analysis of true combinatorial libraries

Combinatorial libraries offer new sources of compounds for the research of pharmacological agents such as receptor ligands, enzyme inhibitors or substrates and antibody-binding epitopes. The present review stresses the main roles played by both physico-chemical analysis, particularly when complex mixture of compounds are synthesized as libraries, and biological analysis from which active compounds are identified. After a brief discussion of semantic problems related to the designation of the product mixtures, the physico-chemical analysis of mixtures is reviewed with special emphasis on mass spectrometric techniques. These methods are able both to give a representative view of a library composition and to identify single critical compounds in large libraries. Then the biological screening of such combinatorial libraries is critically discussed with respect to the power and limitations of the methods used for the identification of the active components. Special attention is given to the complex process of library deconvolution. It is pointed out that while combinatorial techniques have evolved towards sophisticated high-tech methods, simple and robust biochemical tests should be used to deconvolute. From a large panel of published examples, a set of trends are identified which should help investigators to choose the most appropriate assay for the discovery of new entities.     

A Linear Relationship between Crystal Size and Fragment Binding Time Observed Crystallographically: Implications for Fragment Library Screening Using Acoustic Droplet Ejection

High throughput screening technologies such as acoustic droplet ejection (ADE) greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handling multiple crystals combined with fragment specimens, these techniques relax the automounter duty-cycle bottleneck that currently prevents optimal exploitation of third generation synchrotrons. Two factors limit the speed and scope of projects that are suitable for fragment screening using techniques such as ADE. Firstly, in applications where the high throughput screening apparatus is located inside the X-ray station (such as the conveyor belt system described above), the speed of data acquisition is limited by the time required for each fragment to soak into its protein crystal. Secondly, in applications where crystals are combined with fragments directly on data acquisition media (including both of the ADE methods described above), the maximum time that fragments have to soak into crystals is limited by evaporative dehydration of the protein crystals during the fragment soak. Here we demonstrate that both of these problems can be minimized by using small crystals, because the soak time required for a fragment hit to attain high occupancy depends approximately linearly on crystal size.     

Discovery of T cell antigens by high-throughput screening of synthetic minigene libraries

The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.