DNA encoding facilitates the construction and screening of large chemical libraries. Here, we describe general strategies for the stepwise coupling of coding DNA fragments to nascent organic molecules throughout individual reaction steps as well as the first implementation of high-throughput sequencing for the identification and relative quantification of the library members. The methodology was exemplified in the construction of a DNA-encoded chemical library containing 4,000 compounds and in the discovery of binders to streptavidin, matrix metalloproteinase 3, and polyclonal human IgG. (5-7), the identification and relative quantification of library members before and after selection can often be achieved by using DNA-microarrays (5-10). By contrast, selections of binding molecules from larger DNA-encoded chemical libraries (comprising several thousand to millions of compounds) may require the use of highthroughput sequencing technologies to assess the relative abundance of library members before and after selection against a target protein of interest.Herein, we describe the construction of a DNA-encoded chemical library consisting of 4,000 compounds covalently attached to unique DNA fragments serving as amplifiable identification bar codes. Similar to our previous experiments with DNA-encoded libraries consisting of several hundreds of members (7), we have initially assessed the relative composition of the new library and its functionality by performing selection experiments on Sepharose resin coated with streptavidin. Because a variety of ligands were known with dissociation constants ranking from the millimolar to the femtomolar range (7) the challenge was to investigate whether binders with various affinities could be easily and rapidly isolated from a library containing 4,000 members. We have found that selections can conveniently be decoded by using a recently described high-throughput DNA sequencing technology (termed ''454 technology'') developed for genome sequencing (11), revealing chemical structures with submicromolar dissociation constants toward streptavidin. In addition, we have performed selections to against the target polyclonal human IgG and the catalytic domain of matrix metalloproteinase 3. To our knowledge a high-throughput sequencing application for decoding of DNA-encoded chemical libraries has not been reported previously. Furthermore, we have devised strategies for the construction and decoding of DNAencoded chemical libraries containing up to 10 6 compounds built on the basis of multiple independent sets of building blocks.
Seeing eye to eye: Plasma‐protein binding is effective in improving the pharmacokinetic properties of otherwise short‐lived molecules. One compound in a class of small portable albumin binders can be used to improve the in vivo circulatory half‐life of two widely used contrast agents. It improves the imaging performance of fluorescein in angiographic analysis of the retina of mice (see picture).
Libraries of chemical compounds individually coupled to encoding DNA tags (DNA‐encoded chemical libraries) hold promise to facilitate exceptionally efficient ligand discovery. We constructed a high‐quality DNA‐encoded chemical library comprising 30 000 drug‐like compounds; this was screened in 170 different affinity capture experiments. High‐throughput sequencing allowed the evaluation of 120 million DNA codes for a systematic analysis of selection strategies and statistically robust identification of binding molecules. Selections performed against the tumor‐associated antigen carbonic anhydrase IX (CA IX) and the pro‐inflammatory cytokine interleukin‐2 (IL‐2) yielded potent inhibitors with exquisite target specificity. The binding mode of the revealed pharmacophore against IL‐2 was confirmed by molecular docking. Our findings suggest that DNA‐encoded chemical libraries allow the facile identification of drug‐like ligands principally to any protein of choice, including molecules capable of disrupting high‐affinity protein–protein interactions.
The identification of specific binding molecules is a central problem in chemistry, biology and medicine. Therefore, technologies, which facilitate ligand discovery, may substantially contribute to a better understanding of biological processes and to drug discovery. DNA-encoded chemical libraries represent a new inexpensive tool for the fast and efficient identification of ligands to target proteins of choice. Such libraries consist of collections of organic molecules, covalently linked to a unique DNA tag serving as an amplifiable identification bar code. DNA-encoding enables the in vitro selection of ligands by affinity capture at sub-picomolar concentrations on virtually any target protein of interest, in analogy to established selection methodologies like antibody phage display. Multiple strategies have been investigated by several academic and industrial laboratories for the construction of DNA-encoded chemical libraries comprising up to millions of DNA-encoded compounds. The implementation of next generation high-throughput sequencing enabled the rapid identification of binding molecules from DNA-encoded libraries of unprecedented size. This article reviews the development of DNA-encoded library technology and its evolution into a novel drug discovery tool, commenting on challenges, perspectives and opportunities for the different experimental approaches.
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