Dual-display of small molecules enables the discovery of ligand pairs and facilitates affinity maturation

Abstract

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.

DNA‐encoded chemical libraries – achievements and remaining challenges

“…After the coupling step, the conjugates can be pooled and split into several vessels, for the reaction with a second set of building blocks (shown in blue). After the reaction step, oligonucleotides can be extended by the use of a splint ligation procedure [21,31], thus incorporating barcodes for the second reaction step (i.e., identifiers for the chemical nature of the individual building blocks used for the second reaction). After a further suitable pool-andsplit procedure, a third set of chemical transformations can be performed (with building blocks depicted in green), thus generating the final compounds, consisting of three sets of building blocks.…”

Acetazolamide Serves as Selective Delivery Vehicle for Dipeptide-Linked Drugs to Renal Cell Carcinoma

“…Unlike antibodies (which can easily be raised against the majority of target proteins of interest), it is not always easy to generate high-affinity small molecule ligands to tumor-associated antigens. However, excellent tumor-targeting results have been reported for folate analogues targeting folate-receptor positive tumors [23,24], substituted urea derivatives targeting prostate-specific membrane antigen [25], somatostatin antagonists targeting the somatostatin receptor [26] and for carbonic anhydrase IX (CAIX) ligands [16][17][18]27].…”

A Specific and Covalent JNK‐1 Ligand Selected from an Encoded Self‐Assembling Chemical Library

“…[3] We have recently described a strategy for the encoding of dualpharmacophore libraries (also called "encoded self-assembling chemical libraries", or ESAC libraries), which was compatible with library decoding procedures, based on high-throughput DNA sequencing. [4] ESAC libraries may facilitate the identification of synergistic building blocks, which recognize adjacent pockets on target proteins of interest. These chemical moieties need to be subsequently connected through a suitable chemical linker, in order to display protein binding in the absence of DNA.…”

“…These chemical moieties need to be subsequently connected through a suitable chemical linker, in order to display protein binding in the absence of DNA. [4,5,6] Encoded chemical libraries have previously been used for the discovery of covalent protein binders. For example, Nicolas Winssinger and coworkers previously reported the identification of reversible and irreversible covalent binders of bromodomain, [7] kinases, [8] proteases and phosphatases [9] from both DNA and PNA encoded chemical libraries.…”

“…The code for oligonucleotides in Sublibrary B (carrying a chemical modification at the 3' end) was introduced by a ligation procedure as previously described. [4] These oligonucleotides contained a short abasic site, allowing the hybridization with members of Sublibrary A. A Klenow polymerization step generated the final and fully encoded library, in which each building block pair was unambiguously identified by two oligonucleotide stretches in Sublibrary A members [ Figure 1].…”