Accelerating the Throughput of Affinity Mass Spectrometry-Based Ligand Screening toward a G Protein-Coupled Receptor

Abstract

Affinity mass spectrometry (MS) enables rapid screening of compound mixtures for ligands bound to a specific protein target, yet its current throughput is limited to individually assay pools of 400−2000 compounds. Typical affinity MS screens implemented in pharmaceutical industry laboratories identify putative ligands based on qualitative analysis of compound binding to the target whereas no quantitative information is acquired to discriminate high-and low-affinity ligands in the screening phase. Furthermore, these screens require purification of a stabilized form of the protein target, which poses a great challenge for membrane receptor targets. Here, we describe a new, potentially general affinity MS strategy that allows screening of 20,000 compounds in one pool for highly efficient ligand discovery toward a G protein-coupled receptor (GPCR) target. Quantitative measurement of compound binding to the receptor enables highaffinity ligand selection using both the purified receptor and receptor-embedded cell membranes. This high-throughput, label-free and quantitative affinity MS screen resulted in discovery of three new antagonists of the A 2A adenosine receptor.

G protein-coupled receptors: structure- and function-based drug discovery

“…By comparing the selection of 16 benchmark A 2A R ligands from screening compound pools of 480-mix, 2400-mix, 4800-mix, and 20K-mix, they demonstrated that this accelerated affinity MS screening approach, using either the purified receptor or receptor-expressing cell membranes, allowed detection of most high-affinity A 2A R ligands ( K d or K i <5 µM), and significant reduction of protein consumption and MS instrument time. 275 Three new antagonists for A 2A R were identified as a result. It is likely that the throughput of this method could be further increased to assay close to or above 1 million compounds in one pool.…”

“…It is likely that the throughput of this method could be further increased to assay close to or above 1 million compounds in one pool. 275 …”

“…More recently, the same team devised another affinity MS strategy that enabled screening of 20,000 compounds in one pool. 275 Specifically, they modified the workflow by performing iterative rounds of affinity selection for compounds associated 266 and Zhang et al 282 with A 2A R. Similar to the previously described single-round affinity MS screening assay, quantitative measurement of BI renders detection of high-affinity ligands in this experiment. By comparing the selection of 16 benchmark A 2A R ligands from screening compound pools of 480-mix, 2400-mix, 4800-mix, and 20K-mix, they demonstrated that this accelerated affinity MS screening approach, using either the purified receptor or receptorexpressing cell membranes, allowed detection of most highaffinity A 2A R ligands (K d or K i <5 µM), and significant reduction of protein consumption and MS instrument time.…”

“…It is likely that the throughput of this method could be further increased to assay close to or above 1 million compounds in one pool. 275 The affinity MS technique has been widely employed to fish out and identify putative ligands toward various enzyme targets from complex extracts of natural products, which could promote lead discovery from TCM. [276][277][278][279][280][281] Indeed, this technique was successfully extended to GPCR ligand screening from herbal extracts.…”

Label‐free Bio‐affinity Mass Spectrometry for Screening and Locating Bioactive Molecules

“…This work connects MNP with GPCRs in the cell membrane for the first time and highlights its unique function in exploiting bioactive compounds. Lu et al (2019) immobilized the purified receptor, A 2A R or HCAR 2 on nickel agarose beads and coupled them with Q Exactive HF LC‐MS, which enables screening of 20,000 compounds in one pool. Nine new chemical modulators for A 2A R have been discovered.…”