Methods for the rapid and inexpensive discovery of hit compounds are essential for pharmaceutical research and DNA-encoded chemical libraries represent promising tools for this purpose. We here report on the design and synthesis of DAL-100K, a DNA-encoded chemical library containing 103 200 structurally compact compounds. Affinity screening experiments and DNA-sequencing analysis provided ligands with nanomolar affinities to several proteins, including prostate-specific membrane antigen and tankyrase 1. Correlations of sequence counts with binding affinities and potencies of enzyme inhibition were observed and enabled the identification of structural features critical for activity. These results indicate that libraries of this type represent a useful source of small-molecule binders for target proteins of pharmaceutical interest and information on structural features important for binding.

DNA-encoded chemical libraries (DELs) represent a versatile and powerful technology platform for the discovery of small-molecule ligands to protein targets of biological and pharmaceutical interest. DELs are collections of molecules, individually coupled to distinctive DNA tags serving as amplifiable identification barcodes. Thanks to advances in DNA-compatible reactions, selection methodologies, next-generation sequencing, and data analysis, DEL technology allows the construction and screening of libraries of unprecedented size, which has led to the discovery of highly potent ligands, some of which have progressed to clinical trials. In this Review, we present an overview of diverse approaches for the generation and screening of DEL molecular repertoires. Recent success stories are described, detailing how novel ligands were isolated from DEL screening campaigns and were further optimized by medicinal chemistry. The goal of the Review is to capture some of the most recent developments in the field, while also elaborating on future challenges to further improve DEL technology as a therapeutic discovery platform.

DNA-encoded combinatorial libraries are increasingly being used as tools for the discovery of small organic binding molecules to proteins of biological or pharmaceutical interest. In the majority of cases, synthetic procedures for the formation of DNA-encoded combinatorial libraries incorporate at least one step of amide bond formation between amino modified DNA and a carboxylic acid. We investigated reaction conditions and established a methodology by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-hydroxy-7-azabenzotriazole and N,N′-diisopropylethylamine (EDC/HOAt/DIPEA) in combination, which provided conversions greater than 75% for 423/543 (78%) of the carboxylic acids tested. These reaction conditions were efficient with a variety of primary and secondary amines, as well as with various types of aminomodified oligonucleotides. The reaction conditions, which also worked efficiently over a broad range of DNA concentrations and reaction scales, should facilitate the synthesis of novel DNAencoded combinatorial libraries. KeywordsDNA-encoded chemical libraries; amide bond formation; synthesis; bioconjugation DNA-encoded combinatorial libraries (DECLs) are collections of organic compounds, individually coupled to distinct DNA fragments, serving as identification barcodes.1-4 Since DNA can be efficiently amplified by the polymerase chain reaction (PCR) and read by highthroughput DNA sequencing methods, the encoding of combinatorial libraries with DNA barcodes allows both the facile identification of specific ligands to protein targets immobilized on a solid support and the convenient handling of the libraries as mixtures of compounds. Compared to High-Throughput Screening, which relies on expensive compound Various chemical strategies have been developed for the generation of large combinatorial DECLs, including DNA-templated synthesis,10,11 DNA-encoded routing,12 hybridizationbased chemical assembling 13-17and DNA-encoded solid-phase synthesis.18 In addition, the iterative assembly of sets of building blocks in a "split-and-pool" methodology, accompanied by the stepwise addition of DNA barcodes (which unambiguously identify the chemical identity of the nascent molecules), is the most generally used strategy for DECLs construction. This strategy facilitates the preparation of DECLs containing millions or even billions of compounds, starting from just few hundred building blocks and oligonucleotides. 19,20,21 In the majority of DECLs disclosed so far, at least one synthesis step involved the formation of an amide bond.4 More than 20'000 carboxylic acids can be purchased from commercial sources at moderate costs (less than $150/g). 22 The acylation of DNA-attached amines with carboxylic acids can be performed on protected DNA structures linked to Controlled Pore Glass (CPG) supports 6 or in solution, 23 followed by deprotection and HPLC purification of the individual conjugates at early stages of library construction. Additionally, for unprotected DNA-attached amines, acylation can be performed either...

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We describe the development of OncoFAP, an ultra-high-affinity ligand of fibroblast activation protein (FAP) for targeting applications with pan-tumoral potential. OncoFAP binds to human FAP with affinity in the subnanomolar concentration range and cross-reacts with the murine isoform of the protein. We generated various fluorescent and radiolabeled derivatives of OncoFAP in order to study biodistribution properties and tumor-targeting performance in preclinical models. Fluorescent derivatives selectively localized in FAP-positive tumors implanted in nude mice with a rapid and homogeneous penetration within the neoplastic tissue. Quantitative in vivo biodistribution studies with a lutetium-177–labeled derivative of OncoFAP revealed a preferential localization in tumors at doses of up to 1,000 nmol/kg. More than 30% of the injected dose had already accumulated in 1 g of tumor 10 min after intravenous injection and persisted for at least 3 h with excellent tumor-to-organ ratios. OncoFAP also served as a modular component for the generation of nonradioactive therapeutic products. A fluorescein conjugate mediated a potent and FAP-dependent tumor cell killing activity in combination with chimeric antigen receptor (CAR) T cells specific to fluorescein. Similarly, a conjugate of OncoFAP with the monomethyl auristatin E-based Vedotin payload was well tolerated and cured tumor-bearing mice in combination with a clinical-stage antibody-interleukin-2 fusion. Collectively, these data support the development of OncoFAP-based products for tumor-targeting applications in patients with cancer.

DNA-encoded chemical libraries are collections of small molecules, attached to DNA fragments serving as identification barcodes, which can be screened against multiple protein targets, thus facilitating the drug discovery process. The preparation of large DNA-encoded chemical libraries crucially depends on the availability of robust synthetic methods, which enable the efficient conjugation to oligonucleotides of structurally diverse building blocks, sharing a common reactive group. Reactions of DNA derivatives with amines and/or carboxylic acids are particularly attractive for the synthesis of encoded libraries, in view of the very large number of building blocks that are commercially available. However, systematic studies on these reactions in the presence of DNA have not been reported so far. We first investigated conditions for the coupling of primary amines to oligonucleotides, using either a nucleophilic attack on chloroacetamide derivatives or a reductive amination on aldehyde-modified DNA. While both methods could be used for the production of secondary amines, the reductive amination approach was generally associated with higher yields and better purity. In a second endeavor, we optimized conditions for the coupling of a diverse set of 501 carboxylic acids to DNA derivatives, carrying primary and secondary amine functions. The coupling efficiency was generally higher for primary amines, compared to secondary amine substituents, but varied considerably depending on the structure of the acids and on the synthetic methods used. Optimal reaction conditions could be found for certain sets of compounds (with conversions >80%), but multiple reaction schemes are needed when assembling large libraries with highly diverse building blocks. The reactions and experimental conditions presented in this article should facilitate the synthesis of future DNA-encoded chemical libraries, while outlining the synthetic challenges that remain to be overcome.

We describe the synthesis and screening of a DNA-encoded chemical library containing 76230 compounds. In this library, sets of amines and carboxylic acids are directly linked producing encoded compounds with compact structures and drug-like properties. Affinity screening of this library yielded inhibitors of the potential pharmaceutical target tankyrase 1, a poly(ADP-ribose) polymerase. These compounds have drug-like characteristics, and the most potent hit compound (X066/Y469) inhibited tankyrase 1 with an IC50 value of 250 nM.

The self-organization of oligopyrene foldamers is described. Bi- and tri-segmental oligomers composed of nucleotides and non-nucleosidic, achiral pyrene monomers form double-stranded helical structures, as shown by absorbance, fluorescence, and CD spectroscopy. The mixed nature of alternating aromatic and phosphate groups ensures water solubility which, in turn, favors folding of the aromatic units. Pyrene molecules also assemble though interstrand stacking interactions. Structural organization of the pyrene units is an intrinsic property of the oligoaryl part and takes place independently from the sequence of the attached DNA. Chirality transfer from DNA to the pyrene segment leads to formation of a double helix, in which neighboring pyrene units are, in the present case, twisted in a right-handed manner. Pyrene helicity is most pronounced in a bi-segmental chimera, in which a DNA stem is present only at one end of the pyrene section.