DNA-Encoded Chemical Libraries: A Comprehensive Review with Succesful Stories and Future Challenges

Abstract

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.

Figure 1

Schematic representations of (A) antibody phage display and (B) DNA-encoded chemical libraries.

Figure 2

Schematic representation describing encoding procedures for DNA-recorded chemical libraries. (A) The small molecules are appended to the dsDNA by a linker (head-piece) and encoded by subsequent DNA ligations using overhang DNA heteroduplexes. (B) Building blocks A and B are encoded by splint ligation, in which the additional DNA fragment is ligated to the nascent DNA structure. Building block C is encoded by the annealing of a partially complementary DNA fragment, followed by a Klenow polymerization procedure.

Figure 3

Schematic representation detailing the encoding strategies for DNA-templated synthesis of chemical libraries. (A) During classical DNA-templated synthesis, short oligonucleotides bearing small-molecule reactants are annealed with the DNA to promote the reaction between the two chemical entities. After the reaction is completed, the linker between the oligonucleotide and the reactant is cleaved and the oligonucleotide is removed by affinity capture. (B) Using a polyinosine segment, universal DNA-templated synthesis avoids the use of high-fidelity annealing between the oligonucleotide promoter and the DNA. (C) The YoctoReactor technology promotes highly efficient intermolecular reactions by close proximity, using 3D DNA junctions. (D) On-resin immobilized DNA complementary codons are used to promote annealing with the different DNA-conjugated small molecules and subsequent reaction and elution.

Figure 4

Encoding strategies for dual-pharmacophore encoded chemical libraries. During encoded self-assembling chemical libraries (ESAC) synthesis, both strands A and B bearing the two distinct chemical moieties are prepared and purified individually, offering an extremely high degree of purity to the final library. Upon annealing between the code A and the d-spacer oligonucleotide, the code B is transferred onto the complementary strand by Klenow polymerization.

Figure 5

Schematic representation of different hi-EDCCL approaches. (A) hi-EDCCLs are based on partial annealing of two complementary sub-libraries. The DNA duplex is unstable until the addition of the target displaces the equilibrium toward the high-affinity combinations. The non-binding moieties can be re-shuffled to identify the best combinations. (B) An evolution of hi-EDCCL is based on the generation of a physical linkage ([2+2] UV-promoted cycloaddition) between both DNA strands once the equilibrium gets shifted upon addition of the target. (C) The latest advancement on EDCCL relies on the formation of Y-shaped DNA constructs to facilitate the dynamic enrichment of synergistic binding pairs.

Figure 6

Schematic representation of selection methods for DNA-encoded chemical libraries. (A) Affinity capture of the desired target using magnetic beads is the most common approach for DEL screenings. Upon immobilization, the library is incubated with the target, and the non-binding molecules are washed away by stringent washing procedures. (B) The use of resin tips avoids the necessity of magnetic capturing of the protein–ligand complexes. Once the target is immobilized in the core of the tip, the library passes through it, and the binding molecules get retained in the matrix. After subsequent heat elution, the binding moieties are recovered. (C) The target of interest is modified to include a DNA tag, which hybridizes with a short complementary region in the library once the ligands approach the protein. Only binder–protein complexes are PCR amplified. (D) Photo-cross-linking methodologies allow the formation of a covalent bond between the binding molecules and the target protein. The photo-cross-linking library is incubated with the target protein, and upon irradiation the ligand–protein complex is fixed. Subsequent stringent washings discard the non-binders.

Figure 7

Non-exhaustive list of validated initial hits from DEL screenings against targets of pharmaceutical relevance.