insight

DNA-encoded chemical libraries (DELs) are useful tools for the discovery of small molecule ligands to protein targets of pharmaceutical interest. Compared with single-pharmacophore DELs, dual-pharmacophore DELs simultaneously display two chemical moieties on both DNA strands, and allow for the construction of highly diverse and pure libraries, with a potential for targeting larger protein surfaces. Although methods for the encoding of simple, fragment-like dual-display libraries have been established, more complex libraries require a different encoding strategy. Here, we present a robust and convenient "large encoding design" (LED), which facilitates the PCR-amplification of multiple codes distributed among two partially complementary DNA strands. We experimentally implemented multiple coding regions and we compared the new DNA encoding scheme with previously reported dual-display DEL modalities in terms of amplifiability and performance in test selections against two target proteins. With the LED methodology in place, we foresee the construction and screening of DELs of unprecedented sizes and designs.

Source: https://doi.org/10.1039/D1SC05721A

Placental alkaline phosphatase (PLAP) is an abundant surface antigen in the malignancies of the female reproductive tract. Nevertheless, the discovery of PLAP-specific small organic ligands for targeting applications has been hindered by ligand cross-reactivity with the ubiquitous tissue non-specific alkaline phosphatase (TNAP). In this study, we used DNA-encoded chemical libraries to discover a potent (IC50 = 32 nM) and selective PLAP inhibitor, with no detectable inhibition of TNAP activity. Subsequently, the PLAP ligand was conjugated to fluorescein; it specifically bound to PLAP-positive tumors in vitro and targeted cervical cancer in vivo in a mouse model of the disease. Ultimately, the fluorescent derivative of the PLAP inhibitor functioned as a bispecific engager redirecting the killing of chimeric antigen receptor-T cells specific to fluorescein on PLAP-positive tumor cells.

Source: https://doi.org/10.1021/acs.jmedchem.1c01103

DNA‐encoded chemical libraries are typically screened against purified protein targets. Recently, cell‐based selections with encoded chemical libraries have been described, commonly revealing suboptimal performance due to insufficient recovery of binding molecules. We used carbonic anhydrase IX (CAIX)‐expressing tumor cells as a model system to optimize selection procedures with code‐specific quantitative polymerase chain reaction (qPCR) as selection readout. Salt concentration and performing PCR on cell suspension had the biggest impact on selection performance, leading to 15‐fold enrichment factors for high‐affinity monovalent CAIX binders (acetazolamide; K D = 8.7 nM). Surprisingly, the homobivalent display of acetazolamide at the extremities of both complementary DNA strands led to a substantial improvement of both ligand recovery and enrichment factors (above 100‐fold). The optimized procedures were used for selections with a DNA‐encoded chemical library comprising 1 million members against tumor cell lines expressing CAIX, leading to a preferential recovery of known and new ligands against this validated tumor‐associated target. This work may facilitate future affinity selections on cells against target proteins which might be difficult to express otherwise.

Source: https://doi.org/10.1002/chem.202100816