Target-specific labeling of membrane proteins for ligand discovery and other applications

Abstract

Membrane proteins perform vital cellular functions in a myriad physiological processes in vivo, implicating in a variety of pathways they are associated with disease pathogenesis and progression. As a result, membrane protein is a major class of drug targets and has exhibited high therapeutic significance in modern medicine. However, drug discovery targeting membrane proteins is extremely challenging due to their hydrophobic nature and large molecular size. Moreover, membrane proteins heavily depend on the natural cell membrane environment to maintain their natural structures and biological functions, which are often required for developing efficacious drug candidates. Hence, novel methods and techniques compatible with native cellular circumstance are highly desired to study and target membrane proteins. As a powerful ligand discovery technology, DNA-encoded chemical library (DEL) has recently emerged and become a prominent approach in discovering novel ligands against biological targets. However, DEL selections are mostly only suitable for immobilized purified protein targets, largely restricting it from further implementation to endogenous membrane proteins. In response, I herein propose a novel cell labeling approach where target membrane proteins on live cells are covalently tagged by single stranded DNA molecules which serve as homing beacons for DEL hybridization, thereby realizing target-specific cell-based selections of large-scale DELs for membrane proteins in their natural cellular habitats. In Chapter 3, the performance of the selection method was first assessed with model systems, before progressing into the selection of a 30.42-million member DEL against representative membrane proteins. A series of novel ligands have been identified from the selections. These studies have validated the generality of the method as well as its potential to facilitate membrane-protein-based drug discovery. In addition, benefiting from the programmability of the DNA tags, the DNA-labeled cells also enable a variety of target-specific applications, including fluorescence-based on-cell ligand screening, cell surface protein detection and imaging, targeted drug delivery, programmed cell assembly, and DNA-directed cell sorting in Chapter 4.