In contrast to standard fragment-based drug discovery approaches, dual-display DNA-encoded chemical libraries have the potential to identify fragment pairs that bind simultaneously and benefit from the chelate effect. However, the technology has been limited by the difficulty in unambiguously decoding the ligand pairs from large combinatorial libraries. Here we report a strategy that overcomes this limitation and enables the efficient identification of ligand pairs that bind to a target protein. Small organic molecules were conjugated to the 5' and 3' ends of complementary DNA strands that contain a unique identifying code. DNA hybridization followed by an inter-strand code-transfer created a stable dual-display DNA-encoded chemical library of 111,100 members. Using this approach we report the discovery of a low micromolar binder to alpha-1-acid glycoprotein and the affinity maturation of a ligand to carbonic anhydrase IX, an established marker of renal cell carcinoma. The newly discovered subnanomolar carbonic anhydrase IX binder dramatically improved tumour targeting performance in vivo.

The isolation of molecules capable of high-affinity and specific binding to biological targets is a central problem in chemistry, biology and pharmaceutical sciences. Here we describe the use of encoded self-assembling chemical (ESAC) libraries for the facile identification of molecules that bind macromolecular targets. ESAC technology uses libraries of organic molecules linked to individual oligonucleotides that mediate the self-assembly of the library and provide a code associated with each organic molecule. After panning ESAC libraries on the biomolecular target of interest, the 'binding code' of the selected compounds can be 'decoded' by a number of experimental techniques (e.g., hybridization on oligonucleotide microarrays). The potential of this technology was demonstrated by the affinity maturation (>40-fold) of binding molecules to human serum albumin and bovine carbonic anhydrase, leading to binders with dissociation constants in the nanomolar range.

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).

DNA-encoded chemical libraries (DECLs) represent a promising tool in drug discovery. DECL technology allows the synthesis and screening of chemical libraries of unprecedented size at moderate costs. In analogy to phage-display technology, where large antibody libraries are displayed on the surface of filamentous phage and are genetically encoded in the phage genome, DECLs feature the display of individual small organic chemical moieties on DNA fragments serving as amplifiable identification barcodes. The DNA-tag facilitates the synthesis and allows the simultaneous screening of very large sets of compounds (up to billions of molecules), because the hit compounds can easily be identified and quantified by PCR-amplification of the DNA-barcode followed by high-throughput DNA sequencing. Several approaches have been used to generate DECLs, differing both in the methods used for library encoding and for the combinatorial assembly of chemical moieties. For example, DECLs can be used for fragment-based drug discovery, displaying a single molecule on DNA or two chemical moieties at the extremities of complementary DNA strands. DECLs can vary substantially in the chemical structures and the library size. While ultralarge libraries containing billions of compounds have been reported containing four or more sets of building blocks, also smaller libraries have been shown to be efficient for ligand discovery. In general, it has been found that the overall library size is a poor predictor for library performance and that the number and diversity of the building blocks are rather important indicators. Smaller libraries consisting of two to three sets of building blocks better fulfill the criteria of drug-likeness and often have higher quality. In this Account, we present advances in the DECL field from proof-of-principle studies to practical applications for drug discovery, both in industry and in academia. DECL technology can yield specific binders to a variety of target proteins and is likely to become a standard tool for pharmaceutical hit discovery, lead expansion, and Chemical Biology research. The introduction of new methodologies for library encoding and for compound synthesis in the presence of DNA is an exciting research field and will crucially contribute to the performance and the propagation of the technology.