Primer Extension-Mediated Aptamer-Gated DNA-Encoded Library Screening for Physiologically Relevant Ligand Discovery.

Area of Science:

• Biochemistry
• Drug Discovery
• Molecular Biology

Background:

• DNA-encoded library (DEL) technologies are powerful tools in drug discovery.
• Current DEL methods often use purified targets, limiting the physiological relevance of discovered ligands.
• There is a need for DEL platforms that can screen in more native-like environments.

Purpose of the Study:

• To introduce and validate the Primer Extension-Mediated Aptamer-Gated DEL Screening (PEMAG-DEL) platform.
• To enable ligand discovery in complex, native-like biological environments.
• To identify high-affinity ligands for carbonic anhydrase II (CA II) using the PEMAG-DEL platform.

Main Methods:

• Development of the PEMAG-DEL platform integrating aptamer-guided target localization, enzymatic filtering, and combinatorial chemistry.
• Design and synthesis of a 125-member DNA-encoded library of peptide-sulfonamide conjugates.
• Screening and selection of ligands against carbonic anhydrase II (CA II) in both purified and cell lysate conditions.

Main Results:

• Identification of a high-affinity ligand, Sulfo-His-His-Ala, with a 16.3-fold improved binding affinity (IC50 = 0.84 μM) compared to the parent compound.
• Demonstration of PEMAG-DEL's robustness against nonspecific interference from membrane proteins and aggregates in HeLa cell lysates.
• Successful selection of a biologically relevant ligand using the novel platform in a complex cellular environment.

Conclusions:

• The PEMAG-DEL platform enables precise ligand selection in native-like, complex biological environments.
• PEMAG-DEL overcomes limitations of traditional DEL screening by reducing nonspecific interference.
• This technology holds significant promise for discovering physiologically relevant drug candidates.