Lead compounds discovered from libraries: part 2

Many lead compounds with the potential to progress to viable drug candidates have been identified from libraries during the past two years. There are two key strategies most often employed to find leads from libraries: first, high-throughput biological screening of corporate compound collections; and second, synthesis and screening of project-directed libraries (i.e. target-based libraries). Numerous success stories, including the discovery of several clinical candidates, testify to the utility of chemical library collections as proven sources of new leads for drug development.     

Modern methods to produce natural-product libraries

Natural sources offer a wealth of chemically diverse compounds that have been evolutionary preselected to modulate biochemical pathways. Several industrial and academic groups are accessing this source using advanced technology platforms. Methods have been reported to generate large and diverse natural-product libraries optimised for high-throughput screening and for a fast discovery process. In addition to prefractionated and pure natural-product libraries, parallel synthesis gives access to synthetic, semi-synthetic and natural-product-like libraries. Natural-product chemistry and organic synthesis are powerful tools for optimising natural leads and for generating new diversity from natural scaffolds. The amalgamation of both may be expected to become an important strategy in future drug design.     

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.     

Chromosome specific c-DNA libraries: reduction of unspecific priming events by purification of heteronuclear RNA

Chromosome specific c-DNA libraries greatly facilitate the isolation of disease associated genes which have been previously linked to particular chromosomes. Recently, several methods have been developed and employed for the isolation of transcribed sequences from specific human chromosomes and chromosome regions. Heteronuclear (hn) RNA from somatic human/rodent cell hybrids has been used as starting material to selectively prime the synthesis of human specific c-DNAs. A drawback of this method is the high number of rodent clones found in these chromosome specific c-DNA libraries. Here, we provide direct evidence that unspecific priming events account for the majority of these rodent clones. Using an Alu consensus primer hn-RNA human specific c-DNA libraries have been established and the specificity of Alu-priming has been evaluated. Using a variety of purification schemes for isolating hn-RNA we have significantly reduced the percentage of unspecific priming events. We also included a comparison of the hn-RNA yield from different somatic hybrids prior and after purification.     

Natural product-like libraries based on non-aromatic, polycyclic motifs

Diversity-oriented synthesis is an intriguing approach for creating structurally diverse compounds that cover the pharmaceutically relevant chemical space in an optimal way. On the other hand, an over-proportionally large number of drugs or lead structures originate from compounds isolated from natural sources. Thus, not surprisingly, an increasing number of combinatorial libraries are based on motifs resembling natural products. A particular aspect of many natural products is the presence of non-aromatic, polycyclic core structures. The fusion of several rings leads to geometrically well-defined structures and, thus, holds the promise of a high functional specialisation. In this review we present several actual examples of natural product-like libraries with non-aromatic polycyclic motifs based on naturally occurring compounds.     

Combinatorial catalyst discovery.

There have been recent attempts to use the principles of combinatorial chemistry and high-throughput screening strategies for catalyst identification. With the technology available that allows the synthesis of large libraries, scientists of varied backgrounds have implemented screening efforts to identify active and selective catalysts. Within this context, several techniques have come to light in the past year: infrared thermography is used to identify optimal catalysts by monitoring the change in temperature for exothermic reactions; fluorescence and colored-dye assays, a familiar tool to biologists, is being applied to the identification of catalysts that exhibit the highest activity. Whereas none of these screening methods provide a general solution to the problem of screening large combinatorial libraries (there is likely to be no general solution), each advance represents an important intellectual and technological step forward.     

Libraries of non-polymeric organic molecules

New technology is emerging that permits the chemical synthesis of large numbers of different compounds simultaneously. Combinatorial chemistry is heavily dependent upon the adaption of organic synthesis to solid supports and has necessitated the development of appropriate analytical and chemical approaches to both monitor solid-phase reactions and release finished compounds into solution. Considerable progress has recently been made in all of these areas. Small-molecule libraries of medicinally important chemical classes, such as 1,4-benzodiazepines, mercaptopropionyl amino acids, and peptidyl phosphonates, have recently been reported. Encoded combinatorial libraries of dihydrobenzopyran-based and acylpiperidine-based pharmacophores have yielded potent inhibitors of carbonic anhydrase. Automated instrumentation is growing in importance for the synthesis of small-molecule libraries.     

An improved method of partially digesting plant megabase DNA suitable for YAC cloning: application to the construction of a 5.5 genome equivalent YAC library of tomato

An improved method for preparing partially digested tomato DNA has been developed, that is suitable for YAC cloning. It involves (i) isolation of high molecular-weight DNA from agarose-embedded leaf protoplasts, (ii) controlled partial digestion in situ using Eco RI endonuclease in the presence of Eco RI methylase (M. Eco RI), and (iii) fractionation of the partial digest on a Clamped Homogeneous Electric Fields (CHEF) gel. Unlike methods commonly used for generating partial digests, the present method allows one to produce digests in which the bulk of restriction fragments are of the desired size. Use of these partial digests in constructing YAC libraries of the tomato lines Moneymaker- Cf4 and VFNT Cherry resulted in libraries (total 21 060 clones, 5.5 genome equivalents) in which 80% of the YACs have inserts between 200 and 600 kb. Both libraries have been screened with selected RFLP markers linked to the Cladosporium fulvum Cf4 locus on chromosome 1, using a three-dimensional PCR-based screening technique. To this end, the RFLP markers have been sequenced to allow for the synthesis of specific primers. Thus, for each marker tested several YAC clones have been isolated, including a family of clones that carry leucine-rich repeat sequences located around the Cf4/ Cf9 locus.     

Incremental truncation as a strategy in the engineering of novel biocatalysts

The application and success of combinatorial approaches to protein engineering problems have increased dramatically. However, current directed evolution strategies lack a combinatorial methodology for creating libraries of hybrid enzymes which lack high homology or for creating libraries of highly homologous genes with fusions at regions of non-identity. To create such hybrid enzyme libraries, we have developed a series of combinatorial approaches that utilize the incremental truncation of genes, gene fragments or gene libraries. For incremental truncation, Exonuclease III is used to create a library of all possible single base-pair deletions of a given piece of DNA. Incremental truncation libraries (ITLs) have applications in protein engineering as well as protein folding, enzyme evolution, and the chemical synthesis of proteins. In addition, we are developing a methodology of DNA shuffling which is independent of DNA sequence homology.     

Design, synthesis, and biological evaluation of abiotic, non-lactone modulators of LuxR-type quorum sensing

Quorum sensing (QS) is a cell-cell signaling mechanism that allows bacteria to monitor their population size and alter their behavior at high cell densities. Gram-negative bacteria use N-acylated L-homoserine lactones (AHLs) as their primary signals for QS. These signals are susceptible to lactone hydrolysis in biologically relevant media, and the ring-opened products are inactive QS signals. We have previously identified a range of non-native AHLs capable of strongly agonizing and antagonizing QS in Gram-negative bacteria. However, these abiotic AHLs are also prone to hydrolysis and inactivation and thereby have a relatively short time window for use (～12-48 h). Non-native QS modulators with reduced or no hydrolytic instability could have enhanced potencies and would be valuable as tools to study the mechanisms of QS in a range of environments (for example, on eukaryotic hosts). This study reports the design and synthesis of two libraries of new, non-hydrolyzable AHL mimics. The libraries were screened for QS modulatory activity using LasR, LuxR, and TraR bacterial reporter strains, and several new, abiotic agonists and antagonists of these receptors were identified.     

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides¹. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously¹. This combinatorial platform has been validated with conventional methods² and the polyanhydride film and nanoparticle libraries have been characterized with ¹H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion^1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.     

Design of compound libraries based on natural product scaffolds and protein structure similarity clustering (PSSC)

Recent advances in structural biology, bioinformatics and combinatorial chemistry have significantly impacted the discovery of small molecules that modulate protein functions. Natural products which have evolved to bind to proteins may serve as biologically validated starting points for the design of focused libraries that might provide protein ligands with enhanced quality and probability. The combined application of natural product derived scaffolds with a new approach that clusters proteins according to structural similarity of their ligand sensing cores provides a new principle for the design and synthesis of such libraries. This article discusses recent advances in the synthesis of natural product inspired compound collections and the application of protein structure similarity clustering for the development of such libraries.     

Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development.     

Synthetic methods of glycopeptide assembly, and biological analysis of glycopeptide products

The technology of glycopeptide synthesis has recently developed into a fully mature science capable of creating diverse glycopeptides of biological interest, even in combinatorial displays. This has allowed biochemists to investigate substrate specificity in the biosynthetic processing and immunology of various protein glycoforms. The construction of all the mucin core structures and a varietyof cancer-related glycopeptides has facilitated detailed analysis of the interaction between MHC-bound glycopeptides and T cell receptors. Novel dendritic neoglycopeptide ligands have been shown to demonstrate high affinity for carbohydrate receptors and these interactions are highly dendrimer specific. Large complex N-linked oligosaccharides have been introduced into glycopeptides using synthetic or chemoenzymatic procedures, both methods affording pure glycopeptides corresponding to a single glycoform in preparative quantities. The improved availability of glycosyl transferases has led to increased use of chemoenzymatic synthesis. Chemical ligation has been introduced as a method of attaching glycans to peptide templates. Combinatorial synthesis and the analysis of resin-bound glycopeptide libraries have been successfully carried out by applying the ladder synthesis principle. Direct quantitative glycosylation of peptide templates on solid phase has paved the way for the synthesis of templated glycopeptide mixtures as libraries of libraries.     

Solid-phase combinatorial synthesis using MicroKan reactors, Rf tagging, and directed sorting

A modular system for high-output solid-phase combinatorial synthesis has been designed and developed. The system employs three technological innovations to achieve its high efficiency and reliability: (1) application of microreactors as the reaction units in solid-phase synthesis; (2) use of radiofrequency tagging as the non-chemical tracking method; and (3) development of the directed sorting technology for split & pool synthesis. The system has been successfully applied in the synthesis of compound libraries of several hundred to several thousand compounds in multi-milligrams per compound quantity by many organizations.     

Genes expressed in the male gametophyte of flowering plants and their isolation

Recombinant cDNA libraries to poly(A)RNA isolated from mature pollen of Zea mays and Tradescantia paludosa have been constructed. Northern blot analyses indicate that several of the clones are unique to pollen and are not expressed in vegetative tissues. The majority, however, are expressed both in pollen and vegetative tissues. Southern hybridizations show that the pollen specific sequences in corn are present in one or a very few copies in the genome. By using several of the clones as probes, it was found that there are at least two different groups of mRNAs with respect to their synthesis. The mRNAs of the first group represented by the pollen specific clones are synthesized after microspore mitosis and increase in concentration up to maturity. The second group, exemplified by actin mRNA, begins to accumulate soon after meiosis, reaches its maximum by late pollen interphase, and decreases thereafter. Although the actin mRNA and the pollen specific mRNAs studied show very different patterns of initiation of synthesis and accumulation during pollen development, the rates of decline of these mRNAs during the first 60 minutes of germination and pollen tube growth in Tradescantia are similar and reflect the previously observed declines in rates of protein synthesis during this period.     

Combinatorial biocatalysis

The published applications of combinatorial biocatalysis have continued to expand at a growing rate. This is exemplified by the variety of enzyme catalysts and whole-cell catalysts used for the creation of libraries through a wide range of biocatalytic reactions, including acylation, glycosylation, halogenation, oxidation and reduction. These biocatalytic methods add the capability to perform unique chemistries or selective reactions with complex or labile reagents when integrated with classical combinatorial synthesis methods. Thus, applications towards the production of libraries de novo, the expansion of chemically derived combinatorial libraries, and the generation of novel combinatorial reagents for library synthesis can be achieved. Theoretically, these results illustrate what is already evident from nature: that complex, biologically active, structurally diverse compound libraries can be generated through the application of biocatalysis alone or in combination with classical organic synthesis approaches.