A new approach for modification of phenylalanine peptides by Suzuki-Miyaura coupling reaction

For the first time, we have modified phenylalanine peptides by the Suzuki-Miyaura coupling reaction which may be useful in developing combinatorial libraries of peptidomimetics.     

A Convenient Approach to Prepare Topologically Segregated Bilayer Beads for One-Bead Two-Compound Combinatorial Peptide Libraries

One-bead one-compound (OBOC) combinatorial peptide libraries have been used to identify ligands and modulators for a wide variety of biological targets. While being very efficient with linear peptides, OBOC libraries with N-terminally blocked peptides or with unsequenceable building blocks require encoding. To fully exploit OBOC combinatorial methods with cyclic peptides and peptidomimetics, topologically segregated bilayer beads have been developed. This strategy offers the opportunity to synthesize two compounds per bead, i.e. with one compound exposed on the bead surface for screening, and the other one found within the inner layer as a tag for sequencing and compound identification. Bead segregation often involves the use of unstable derivatives or requires a series of protection–deprotection steps. In order to expedite and optimize bead segregation, the performance of various reagents has been studied. The results obtained herein show that bead segregation can be efficiently performed with commercially available reagents. Finally, in order to control outer/inner layer ratios in segregated beads, the effects of different parameters have been evaluated. We report a straightforward and efficient procedure to prepare topologically segregated bilayer beads in a wide range of controllable, predictable, and reproducible outer versus inner ratios.     

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both α-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.     

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.     

DNA assembly techniques for next-generation combinatorial biosynthesis of natural products

Natural product scaffolds remain important leads for pharmaceutical development. However, transforming a natural product into a drug entity often requires derivatization to enhance the compound’s therapeutic properties. A powerful method by which to perform this derivatization is combinatorial biosynthesis, the manipulation of the genes in the corresponding pathway to divert synthesis towards novel derivatives. While these manipulations have traditionally been carried out via restriction digestion/ligation-based cloning, the shortcomings of such techniques limit their throughput and thus the scope of corresponding combinatorial biosynthesis experiments. In the burgeoning field of synthetic biology, the demand for facile DNA assembly techniques has promoted the development of a host of novel DNA assembly strategies. Here we describe the advantages of these recently developed tools for rapid, efficient synthesis of large DNA constructs. We also discuss their potential to facilitate the simultaneous assembly of complete libraries of natural product biosynthetic pathways, ushering in the next generation of combinatorial biosynthesis.     

Structure-based library design: molecular modelling merges with combinatorial chemistry

Recent advances in both computational and experimental techniques now allow a very fruitful interplay of computational and combinatorial chemistry in the structure-based design of combinatorial libraries.     

Diversity-oriented synthesis: exploring the intersections between chemistry and biology

Diversity-oriented synthesis (DOS) is an emerging field involving the synthesis of combinatorial libraries of diverse small molecules for biological screening. Rather than being directed toward a single biological target, DOS libraries can be used to identify new ligands for a variety of targets. Several different strategies for library design have been developed to target the biologically relevant regions of chemical structure space. DOS has provided powerful probes to investigate biological mechanisms and also served as a new driving force for advancing synthetic organic chemistry.     

Novel miniaturized systems in high-throughput screening

High-throughput screening (HTS) using high-density microplates is the primary method for the discovery of novel lead candidate molecules. However, new strategies that eschew 2D microplate technology, including technologies that enable mass screening of targets against large combinatorial libraries, have the potential to greatly increase throughput and decrease unit cost. This review presents an overview of state-of-the-art microplate-based HTS technology and includes a discussion of emerging miniaturized systems for HTS. We focus on new methods of encoding combinatorial libraries that promise throughputs of as many as 100,000 compounds per second.     

Dissimilarity-based algorithms for selecting structurally diverse sets of compounds.

This paper commences with a brief introduction to modern techniques for the computational analysis of molecular diversity and the design of combinatorial libraries. It then reviews dissimilarity-based algorithms for the selection of structurally diverse sets of compounds in chemical databases. Procedures are described for selecting a diverse subset of an entire database, and for selecting diverse combinatorial libraries using both reagent-based and product-based selection.     

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.     

Comparison of mRNA-display-based selections using synthetic peptide and natural protein libraries

mRNA display is a genotype-phenotype conjugation method that allows the amplification-based, iterative rounds of in vitro selection to be applied to peptides and proteins. Compared to prior protein selection techniques, mRNA display can be used to select functional sequences from both long natural protein and short combinatorial peptide libraries with much higher complexities. To investigate the basic features and problems of using mRNA display in studying conditional protein-protein interactions, we compared the target-binding selections against calmodulin (CaM) using both a natural protein library and a combinatorial peptide library. The selections were efficient in both cases and required only two rounds to isolate numerous Ca2+/CaM-binding natural proteins and synthetic peptides with a wide range of affinities. Many known and novel CaM-binding proteins were identified from the natural human protein library. More than 2000 CaM-binding peptides were selected from the combinatorial peptide library. Unlike sequences from prior CaM-binding selections that correlated poorly with naturally occurring proteins, synthetic peptides homologous to the Ca2+/CaM-binding motifs in natural proteins were isolated. Interestingly, a large number of synthetic peptides that lack the conventional CaM-binding secondary structures bound to CaM tightly and specifically, suggesting the presence of other interaction modes between CaM and its downstream binding targets. Our results indicate that mRNA display is an ideal approach to the identification of Ca2+-dependent protein-protein interactions, which are important in the regulation of numerous signaling pathways.     

From Combinatorial Libraries to MHC Ligand Motifs, T-cell Superagonists and Antagonists

Complete experimental data sets of HLA-ligand motifs and T-cell recognition patterns can be derived from combinatorial peptide libraries. These data provide the exact molecular basis for a fast development of synthetic vaccines, T-cell superagonists and non-peptide antagonists. Patient-specific peptides, peptidomimetics and vaccines of highest reactivity can be derived directly from the data sets via our prediction programme EPIPREDICT. The resulting lead structures may be developed into valuable diagnostics and therapeutic tools for the treatment of viral infections, autoimmune diseases and tumors. As one example, antibody and T cell recognition in the intestinal auto-immune disease, coeliac disease was investigated in more detail concerning the deamidation of gamma-gliadin peptides by tissue transglutaminase 9tTG) leading to autoreactive peptides specific for HLA-DQA1*0501, DQB1*0201.     

Unnatural RNA display libraries

Combinatorial peptide and protein libraries have now been developed to accommodate unnatural amino acids in a genetically encoded format via in vitro nonsense and sense suppression. General translation features and specific regioselective and stereoselective properties of the ribosome endow these libraries with a broad chemical diversity. Alternatively, amino acid residues can be chemically derivatized post-translationally to add preferred functionality to the encoded peptide. All of these efforts are advancing combinatorial peptide and protein libraries for enhanced ligands against biological targets of interest.     

Peptide phage display in biotechnology and biomedicine

To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors, etc.). Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nanomaterials.     

Solid phase synthesis of complex natural products and libraries thereof.

Natural products have served as an important source of medicinal compounds and pharmaceutical leads over the last century. Within the last 10 years, significant interest has developed in applying combinatorial chemistry techniques to the study of natural products and their biological activities. In this review, we examine several representative efforts wherein natural product skeletons have been constructed or immobilized on solid support and subsequently derivatized, giving rise to analog libraries useful in understanding the structure-activity relationships of the parent natural product. Issues such as target selection, library design, linker development, automation, and library characterization are addressed.     

A Combinatorial Approach for the Design of Complementarity-determining Region-derived Peptidomimetics with in Vitro Anti-tumoral Activity

The great success of therapeutic monoclonal antibodies has fueled research toward mimicry of their binding sites and the development of new strategies for peptide-based mimetics production. Here, we describe a new combinatorial approach for the production of peptidomimetics using the complementarity-determining regions (CDRs) from gastrin17 (pyroEGPWLEEEEEAYGWMDF-NH₂) antibodies as starting material for cyclic peptide synthesis in a microarray format. Gastrin17 is a trophic factor in gastrointestinal tumors, including pancreatic cancer, which makes it an interesting target for development of therapeutic antibodies. Screening of microarrays containing bicyclic peptidomimetics identified a high number of gastrin binders. A strong correlation was observed between gastrin binding and overall charge of the peptidomimetic. Most of the best gastrin binders proceeded from CDRs containing charged residues. In contrast, CDRs from high affinity antibodies containing mostly neutral residues failed to yield good binders. Our experiments revealed essential differences in the mode of antigen binding between CDR-derived peptidomimetics (K_d values in micromolar range) and the parental monoclonal antibodies (K_d values in nanomolar range). However, chemically derived peptidomimetics from gastrin binders were very effective in gastrin neutralization studies using cell-based assays, yielding a neutralizing activity in pancreatic tumoral cell lines comparable with that of gastrin-specific monoclonal antibodies. These data support the use of combinatorial CDR-peptide microarrays as a tool for the development of a new generation of chemically synthesized cyclic peptidomimetics with functional activity.     

Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution

Directed molecular evolution and combinatorial methodologies are playing an increasingly important role in the field of protein engineering. The general approach of generating a library of partially randomized genes, expressing the gene library to generate the proteins the library encodes and then screening the proteins for improved or modified characteristics has successfully been applied in the areas of protein–ligand binding, improving protein stability and modifying enzyme selectivity. A wide range of techniques are now available for generating gene libraries with different characteristics. This review will discuss these different methodologies, their accessibility and applicability to non-expert laboratories and the characteristics of the libraries they produce. The aim is to provide an up to date resource to allow groups interested in using directed evolution to identify the most appropriate methods for their purposes and to guide those moving on from initial experiments to more ambitious targets in the selection of library construction techniques. References are provided to original methodology papers and other recent examples from the primary literature that provide details of experimental methods.