Development of novel high-throughput biochemical competition assays to identify ligands of human asialoglycoprotein receptor 1

Hepatocyte-specific Asialoglycoprotein receptor (ASGPR) and its native ligand N-acetylgalactosamine (GalNAc) have been actively exploited for targeted delivery of therapeutic and diagnostic agents to the liver. Identification of new potent ligands of ASGPR is of high interest to advance this field and expand to new applications in drug discovery. However, success of novel potent ASGPR ligand discovery has been limited due to the lack of robust high-throughput assays amenable to High-Throughput Screening (HTS). Here, we describe the design and development of two novel biochemical competition binding assays using recombinant human trimeric ASGR1 protein (ASGPR subunit 1) as a mimic of the native multimeric complex and a reference Alexa-647 fluorophore-labelled tri-GalNAc ligand as a tracer. Both ASGR1 TR-FRET and fluorescence polarization (FP) assays are in 384-well microplate format and have a large detection range (IC₅₀ of 2.5 nM - 100 µM), suitable for both monovalent and multivalent ASGPR ligands as well as oligonucleotide conjugates. The ASGR1 FP assay was miniaturized into a 1536-well assay format and a pilot screen of a small molecule library of about 7500 compounds was conducted, identifying 23 positive hits with IC₅₀ values between 12 - 100 µM. Five of the primary hits were validated in orthogonal TR-FRET and SPR binding assays and one of them was successfully docked into the ASGPR, with the docking pose closely matching the binding mode of structurally analogous compound found to be co-crystalized with ASGR1. The successful development of these new ASGR1 biochemical assays provides a new platform for an HTS campaign on small molecule collections to discover novel ASGPR ligands for liver-targeted delivery of efficient therapeutic agents, LYTACs or as potential drugs.