Microchip-based high-throughput screening analysis of combinatorial libraries

In recent years, there have been significant advances in biochemical assay miniturization and integration of microchip-based technologies with combinatorial library screening for high-throughput and large-scale applications. Small-molecule microarrays, protein arrays and cell-based arrays and conventional DNA arrays as well as microfluidic approaches in HTS are discussed in this review.     

High-diversity combinatorial libraries

The synthesis of complex chemical structures by combinatorial chemistry has gained considerable interest. New chemical methods have been developed that enable the synthesis of compound libraries exhibiting structural diversities similar to those of natural products. The concept of 'chemical genomics' has been introduced, reflecting a new quality of understanding and creating the relationship between diverse artificial chemical structures with the space of biological responses and possible protein ligands.     

Genetic optimization of combinatorial libraries

Most agrochemical and pharmaceutical companies have set up high-throughput screening programs which require large numbers of compounds to screen. Combinatorial libraries provide an attractive way to deliver these compounds. A single combinatorial library with four variable positions can yield more than 10(12) potential compounds, if one assumes that about 1000 reagents are available for each position. This is far more than any high-throughput screening facility can afford to screen. We have proposed a method for iterative compound selection from large databases, which identifies the most active compounds by examining only a small fraction of the database. In this article, we describe the extension of this method to the problem of selecting compounds from large combinatorial libraries. Copyright 1998 John Wiley & Sons, Inc.     

Screening phage-displayed combinatorial peptide libraries

Among the many techniques available to investigators interested in mapping protein-protein interactions is phage display. With a modest amount of effort, time, and cost, one can select peptide ligands to a wide array of targets from phage-display combinatorial peptide libraries. In this article, protocols and examples are provided to guide scientists who wish to identify peptide ligands to their favorite proteins.     

Chemical biology of dynamic combinatorial libraries

Dynamic combinatorial chemistry (DCC) is a recently introduced supramolecular approach to generate libraries of chemical compounds based on reversible exchange processes. The building elements are spontaneously and reversibly assembled to virtually encompass all possible combinations, allowing for simple one-step generation of complex libraries. The method has been applied to a variety of combinatorial systems, ranging from synthetic models to materials science and drug discovery, and enables the establishment of adaptive processes due to the dynamic interchange of the library constituents and its evolution toward the best fit to the target. In particular, it has the potential to become a useful tool in the direct screening of ligands to a chosen receptor without extensive prior knowledge of the site structure, and several biological systems have been targeted. In the vast field of glycoscience, the concept may find special perspective in response to the highly complex nature of carbohydrate-protein interactions. This chapter summarises studies that have been performed using DCC in biological systems, with special emphasis on glycoscience.     

Bicyclic carbohydrate-derived scaffolds for combinatorial libraries

A bicyclic scaffold derived from the natural monosaccharide d-glucose, and possessing several diversity sites, was linked to various resins through the primary (C-6) hydroxyl and decorated on the solid phase: the hydroxyl group at C-4 was functionalized as ester, ether, and carbamate, the amino group in the second cycle (C-3' position) was functionalized as amide, sulfonamide, and ureido- and thioureido-derivatives. The compounds synthesized on the solid phase were tested for their antiproliferative activity on tumor cell lines.     

Optical detection methods for combinatorial libraries

The main interests in the development of new combinatorial assays are the reduction of time for screening and an increase in the number of samples measured in parallel. The variety of detection methods is increasing, but the optimal one has not yet been determined. In the past two years, the first parallel detection methods for non-labelled compounds have been developed.     

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.     

Integrating computational and mixture-based screening of combinatorial libraries

Mixture-based synthetic combinatorial library (MB-SCL) screening is a well-established experimental approach for rapidly retrieving structure–activity relationships (SAR) and identifying hits. Virtual screening is also a powerful approach that is increasingly being used in drug discovery programs and has a growing number of successful applications. However, limited efforts have been made to integrate both techniques. To this end, we combined experimental data from a MB-SCL of bicyclic guanidines screened against the κ-opioid receptor and molecular similarity methods. The activity data and similarity analyses were integrated in a biometric analysis–similarity map. Such a map allows the molecules to be categorized as actives, activity cliffs, low similarity to the reference compounds, or missed hits. A compound with IC₅₀ = 309 nM was found in the “missed hits” region, showing that active compounds can be retrieved from a MS-SCL via computational approaches. The strategy presented in this work is general and is envisioned as a general-purpose approach that can be applied to other MB-SCLs.     

Computational design strategies for combinatorial libraries

Medicinal chemistry principles are being increasingly applied to the design of smaller, high purity, information-rich libraries. Recent computational advances in statistical methodology, the design of libraries to reduce ADMET problems, targeting protein families and revisiting natural products as sources of inspiration for scaffolds and reagents are all areas of progressive research.     

Dynamic combinatorial libraries of artificial repeat proteins

Repeat proteins are found in almost all cellular systems, where they are involved in diverse molecular recognition processes. Recent studies have suggested that de novo designed repeat proteins may serve as universal binders, and might potentially be used as practical alternative to antibodies. We describe here a novel chemical methodology for producing small libraries of repeat proteins, and screening in parallel the ligand binding of library members. The first stage of this research involved the total synthesis of a consensus-based three-repeat tetratricopeptide (TPR) protein (～14 kDa), via sequential attachment of the respective peptides. Despite the effectiveness of the synthesis and ligation steps, this method was found to be too demanding for the production of proteins containing variable number of repeats. Additionally, the analysis of binding of the individual proteins was time consuming. Therefore, we designed and prepared novel dynamic combinatorial libraries (DCLs), and show that their equilibration can facilitate the formation of TPR proteins containing up to eight repeating units. Interestingly, equilibration of the library building blocks in the presence of the biologically relevant ligands, Hsp90 and Hsp70, induced their oligomerization into forming more of the proteins with large recognition surfaces. We suggest that this work presents a novel simple and rapid tool for the simultaneous screening of protein mixtures with variable binding surfaces, and for identifying new binders for ligands of interest.     

High affinity extremes in combinatorial libraries and repertoires

By generating a large diversity of molecules, the immune system selects antibodies that bind antigens. Sharing the same approach, combinatorial biotechnologies use a large library of compounds to screen for molecules of high affinity to a given target. Understanding the properties of the best binders in the pool aids the design of the library. In particular, how does the maximum affinity increase with the size of the library or repertoire? We consider two alternative models to examine the properties of extreme affinities. In the first model, affinities are distributed lognormally, while in the second, affinities are determined by the number of matches to a target sequence. The second model more explicitly models nucleic acids (DNA or RNA) and proteins such as antibodies. Using extreme value theory we show that the logarithm of the mean of the highest affinity in a combinatorial library grows linearly with the square root of the log of the library size. When there is an upper bound to affinity, this “absolute maximum” is also approached approximately linearly with root log library size, reaching the upper limit abruptly. The design of libraries may benefit from considering how this plateau is reached as the library size is increased.     

High-throughput characterization and quality control of small-molecule combinatorial libraries

To fully realize the potential of combinatorial synthesis and high-throughput screening for increasing the efficiency of the drug discovery and development process, issues related to compound purity must be addressed. Impurities, often present after synthesis, can lead to ambiguous screening results and inhibit the development of quality structure-activity relationships. The demand for high-throughput analytical characterization of combinatorial libraries has prompted the development of more rapid methods to keep pace with compound production. Recent progress has focused upon the development of parallel separation methods, multiplexed detector interfaces, and synergistic combinations of different detectors possessing complementary selectivities.     

De novo proteins from designed combinatorial libraries

Combinatorial libraries of de novo amino acid sequences can provide a rich source of diversity for the discovery of novel proteins with interesting and important activities. Randomly generated sequences, however, rarely fold into well-ordered proteinlike structures. To enhance the quality of a library, features of rational design must be used to focus sequence diversity into those regions of sequence space that are most likely to yield folded structures. This review describes how focused libraries can be constructed by designing the binary pattern of polar and nonpolar amino acids to favor proteins that contain abundant secondary structure, while simultaneously burying hydrophobic side chains and exposing hydrophilic side chains to solvent. The "binary code" for protein design was used to construct several libraries of de novo proteins, including both alpha-helical and beta-sheet structures. The recently determined solution structure of a binary patterned four-helix bundle is well ordered, thereby demonstrating that sequences that have neither been selected by evolution (in vivo or in vitro) nor designed by computer can form nativelike proteins. Examples are presented demonstrating how binary patterned libraries have successfully produced well-ordered structures, cofactor binding, catalytic activity, self-assembled monolayers, amyloid-like nanofibrils, and protein-based biomaterials.     

Construction of high-complexity combinatorial phage display peptide libraries

Random peptide libraries displayed on the surface of filamentous bacteriophage are widely used as tools for the discovery of ligands for biologically relevant macromolecules, including antibodies, enzymes, and cell surface receptors. Phage display results in linkage of an affinity-selectable function (the displayed peptide) to the DNA encoding that function, allowing selection of individual binding clones by iterative cycles of in vitro panning and in vivo amplification. Critical to the success of a panning experiment is the complexity of the library: the greater the diversity of clones within the library, the more likely the library contains sequences that will bind a given target with useful affinity. A method for construction of high-complexity (> or = 10(9) independent clones) random peptide libraries is presented. The key steps are highly efficient binary ligation under conditions where the vector is relatively dilute, with only a modest molar excess of insert, followed by efficient electrotransformation into Escherichia coli. Library design strategies and a protocol for rapid sequence characterization are also presented.     

Design of combinatorial protein libraries of optimal size

In this article we introduce a computational procedure, OPTCOMB (Optimal Pattern of Tiling for COMBinatorial library design), for designing protein hybrid libraries that optimally balance library size with quality. The proposed procedure is directly applicable to oligonucleotide ligation-based protocols such as GeneReassembly, DHR, SISDC, and many more. Given a set of parental sequences and the size ranges of the parental sequence fragments, OPTCOMB determines the optimal junction points (i.e., crossover positions) and the fragment contributing parental sequences at each one of the junction points. By rationally selecting the junction points and the contributing parental sequences, the number of clashes (i.e., unfavorable interactions) in the library is systematically minimized with the aim of improving the overall library quality. Using OPTCOMB, hybrid libraries containing fragments from three different dihydrofolate reductase sequences (Escherichia coli, Bacillus subtilis, and Lactobacillus casei) are computationally designed. Notably, we find that there exists an optimal library size when both the number of clashes between the fragments composing the library and the average number of clashes per hybrid in the library are minimized. Results reveal that the best library designs typically involve complex tiling patterns of parental segments of unequal size hard to infer without relying on computational means.     

Combinatorial libraries and biological discovery

Combinatorial chemistry has become a popular tool for the preparation of collections of compounds that can be used to find inhibitors and substrates for different protein targets. It has evolved to provide small molecule libraries, which, with the concomittant use of affinity chromatography, gene expression profiling and complementation, can be used to identify compounds and their protein targets in biological systems, including the neurological system.     

Recent developments in the encoding and deconvolution of combinatorial libraries

The value of molecular libraries generated by combinatorial methods is largely dependent on the ease and ability to deconvolute or decode the structure of compounds of interest after screening the library. Following the introduction of promising concepts in the early 1990s, there has been considerable progress in the development and refinement of methodologies to address this issue.