DNA-Encoded Libraries

DNA-encoded libraries (DELs) are now used extensively in both academic and commercial laboratories for the discovery of protein ligands. DEL technology is attractive because it allows very large compound libraries to be screened at a low cost. In this chapter, we consider the origins of DELs in the broader picture of combinatorial chemistry and trace many important developments in the pre-DEL era that shaped current thinking in this area. The strengths and weaknesses of traditional high-throughput screening (HTS), the previously dominant technology for hit finding, are considered. A brief history of the development of both encoded and non-encoded combinatorial libraries is presented, with a particular focus on the critical issue of the “chemical space” covered by the libraries made in the pre-DEL era and how technological limitations shaped their design. We also describe genetically encoded peptide libraries and methods to screen them.

In this chapter, different encoding strategies for DNA- or PNA-encoded chemical libraries are presented. In general, DNA encoding can be distinguished into a DNA-recorded regimen, which encodes the individual chemical reaction steps, and which is most used in practice today, and a DNA-templated regimen where DNA templates guide the chemical synthesis. Various barcoding strategies have been employed most often for the generation of split-and-pool- or DNA-templated single-pharmacophore DELs, but also for dual-pharmacophore DELs displaying fragments/compounds on both ends of a DNA heteroduplex, both in a static and a dynamic setup. In addition to solution-phase protocols, encoded one-bead-one-compound libraries and solid phase-initiated DNA-encoded library synthesis strategies are reported.

DNA-Encoded Libraries (DELs) use chemical reactions to build organic moieties on the coding DNA strand. Accordingly, the generation of a DEL requires robust chemical transformations that are compatible with DNA and the aqueous conditions required for its solubilisation. Reactions that damage DNA cannot be employed, neither can reactions that are hindered by the functionality in DNA (Malone and Paegel, ACS Comb Sci 18(4):182–187, 2016). The nature of DEL synthesis imposes further restrictions: reactions must be compatible with split-and-pool, and reactions must proceed predominantly to the desired product without excessive side products. A broad substrate scope is required using accessible reagents.

As a consequence, a great deal of effort has been expended in developing synthetic methodologies that are DNA-compatible, in order to increase the chemical space DELs can cover. These are, most often, adaptations of off-DNA synthesis methods extended to a DEL setting. The range of reactions available to DEL chemists is ever-expanding, covering an extensive range of reactions including, but not limited to amide couplings, cycloadditions, heterocycle syntheses, nucleophilic additions, reductive aminations, S_NAr reactions, and a wide variety of metal-catalysed cross-couplings, such as Suzuki-Miyaura, Buchwald-Hartwig, and Sonogashira couplings (Kunig et al., Biol Chem 399 (7):691–710, 2018; Shi et al., RSC Adv 11(4):2359–2376, 2021; Castan et al., Bioorg Med Chem 43:116273, 2021; Fair et al., Bioorg Med Chem Lett 51:128339, 2021).

This synthetic toolkit is not all-encompassing; however, new methodologies towards DEL-compatible chemical reactions are constantly being elucidated, broadening the available tools towards DEL synthesis. Herein, recent advances towards DNA-compatible synthetic methods are outlined, comprising DNA damage assessment procedures, technologies to prevent this damage, and applications of these techniques to DEL generation.

Preparing a DNA-encoded chemical library platform is a major undertaking, which requires careful planning. Here we outline general design principles for DNA-encoded libraries on the levels of library topology, chemical reactions, and selection of building blocks. The effects of design parameters on the coverage of the chemical space by a DNA-encoded library and on the properties of encoded compounds are discussed.

The combinatorial nature of DNA-Encoded Libraries (DEL) creates a vast number of compounds covering a wide range of chemistry space, enabling DEL target selections for possible binders that generate enormous amounts of data. This makes advanced cheminformatics and data analysis approaches indispensable for advancing DEL technology for practical applications in the pharmaceutical industry. When designed DEL libraries can be biased to contain compounds with more drug like features through the enumeration of the potential virtual products, based on their combinatorial assembly from available building blocks, and the calculation of molecular descriptors. Computational approaches are also critical in analyzing selection data. In a DEL target selection, it is essential to apply stringent analysis methods to the raw sequencing data to separate real hits from noise and to mitigate the amount of resource required for off-DNA synthesis for hit confirmation. In this chapter we focus on the important role of cheminformatics and computational analysis at all phases of the DEL hit identification workflow by discussing in detail DEL product enumeration, designing DELs of diverse products with focused in specific property spaces, estimating data noise levels in DEL selections, and the process of DEL selection data triage and analysis that eventually identifies hits for on- and off-DNA synthesis. We also outline perspectives of emerging cheminformatics methods for DELs that could further strengthen the technology to facilitate hit identification processes and improve the hit confirmation rate. Emerging methods such as efficient structure searching, visualizing DEL chemistry space, and potential machine learning applications for DEL selection analysis are discussed.

DNA-encoded chemical library (DEL) has emerged as a versatile and innovative technology platform for ligand discovery in chemical biology research and early-stage drug discovery. The rapid development of DEL-compatible reactions has further fueled its applications over the last decade. To date, DELs have been widely adopted by the pharmaceutical industry and are also gaining popularity in academic research. However, a relatively underexplored component of DELs is the selection methodology. DEL selection has been generally considered a massive binding assay, which has been dominated by the so-called bind-wash-elute procedure against immobilized protein targets. Indeed, such a “classical” selection method has seen great success in the selection campaigns against numerous drug targets. Recently, novel DEL selection modalities have emerged, which have not only widened the target scope of DELs to the complex milieu of biological systems but also enabled functional DEL selections beyond identifying physical binders. This chapter furnishes an overview of the current DEL selection methods and concludes with a perspective for future development, aiming to provide a succinct guidance for practitioners who intend to embrace the DEL technology.

DNA-encoded libraries (DELs) have become an increasingly utilized screening technology for identifying chemical matter for drug discovery campaigns. While DELs enable billions of compounds to be screened simultaneously, the combinatorial nature of DELs can often yield hits that fall outside of desired property space; meaning that DEL hit follow-up has typically not followed trends from hits identified from traditional screening techniques that employ highly curated compound collections. This chapter focuses on: (1) important factors to consider when designing and analyzing a DEL screen, (2) follow-up strategies to use once on-DNA hits are identified, (3) analysis of hit-to-lead trends from DEL screens, and (4) case studies of hits that have been developed into clinical candidates.

The history of DNA-encoded chemistry and its evolution into a universally accepted and broadly practised technology for ligand discovery in early stage small-molecule is outlined by a contributor with more than 20 years experience developing the technology. Interviews with the enterpreneures and scientists who made key contributions to DEL technology are included in this personal account of what has been described “In the Pipeline” as “the second coming of combinatorial chemistry, but this time it works!”.