Ligand Binding Assays Beyond GPCRs: Techniques, Best Practices and Applications in Drug Discovery

The pharmaceutical landscape has long been defined by the success of G protein-coupled receptors (GPCRs), which remain the most common target for Food and Drug Administration (FDA)-approved therapeutics. However, as the industry moves toward more complex disease models and the “undruggable” proteome, the focus is expanding to include a diverse array of intracellular and non-canonical proteins. While the list of drugs that target GPCRs continues to grow, the development of non-GPCR-targeting drugs, such as those targeting enzymes, kinases, nuclear receptors, or intracellular proteins, requires a significant shift in analytical methodology. However, successfully navigating these emerging classes requires a transition from traditional membrane-based methodologies to more versatile, sensitive, and physiologically relevant ligand binding assay formats. This article reviews the best techniques and use cases for assays targeting non-GPCR proteins, drawing exclusively from validated methodological frameworks.

Why Explore Ligand Binding Assays Beyond GPCRs?

The scientific community’s historical emphasis on GPCR drug targets stems from their vital role in signal transduction and their accessibility on the cell surface. In fact, GPCR drug target reviews confirm that 36 % of approved drugs modulate GPCR targets, with canonical examples including GLP-1, adrenergic, dopaminergic, and chemokine receptors.

Similar to GPCRs, intracellular proteins are interesting therapeutic targets, both in a traditional occupancy-driven manner as well as a more modern event-driven strategy, because many complex diseases are driven by intracellular pathways and protein-protein interactions that cannot be effectively modulated through classical membrane receptors alone.

They involve new signaling pathways that represent therapeutic opportunities, but detecting interactions in these challenging sites requires highly sensitive protein ligand binding assays capable of identifying low-affinity hits during the early stages of lead discovery. Specifically, these formats should consider:

• Intracellular localization
• Protein conformational flexibility
• Multi-protein complex formation
• Allosteric modulation

In this context, best practices in ligand binding assays become critical to avoid false affinity estimations and mischaracterized mechanisms.

Non-GPCR Drug Targets (Sigma Receptors) and Protein Degradation Pathways (E3 Ligases)

Among emerging non-GPCR drug targets, sigma receptors and E3 ubiquitin ligases stand out for their distinct biology and therapeutic reach.

• Sigma receptors (σRs)

Sigma-1 and Sigma-2 receptors (σ1R and σ2R, respectively) are highly expressed in neuronal tissues and play regulatory roles in calcium signaling, endoplasmic reticulum (ER) stress modulation, and mitochondrial function. Unlike many GPCR targets, sigma-1 acts as a chaperone protein located at the ER-mitochondria interface, influencing cellular homeostasis under stress conditions.

These receptors are under investigation for neurodegenerative and neurodevelopmental disorders such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. Several ligands have shown promising preclinical and early clinical results, positioning sigma receptors as relevant therapeutic nodes beyond conventional GPCR examples.

• Protein degradation pathways (E3 ubiquitin ligases)

E3 ubiquitin ligases are essential mediators for targeted protein degradation (TPD). Rather than inhibiting a protein’s function, TPD strategies direct disease-associated proteins toward proteasomal degradation. Technologies such as PROTACs and PROTABs harness endogenous E3 ligases to degrade intracellular proteins or cell surface receptors. This catalytic mechanism offers several advantages over traditional inhibition:

• Potential to overcome resistance mutations
• Reduced need for sustained occupancy
• Ability to address previously undruggable proteins
• Opportunity for tissue-selective degradation

Recent innovations, including nanobody-based REULR systems, recruit transmembrane E3 ligases to target receptors for degradation, expanding the versatility of degrader platforms.

Figure 1. E3 ligases chemical ligandability, expression patterns, protein-protein interactions (PPI), structure availability, functional essentiality, cellular location, and PPI interface. Adapted from: Liu Y, Yang J, Wang T, Luo M, Chen Y, Chen C, Ronai Z, Zhou Y, Ruppin E, Han L. Expanding PROTACtable genome universe of E3 ligases. Nat Commun. 2023 Oct 16;14(1):6509.

Key Techniques for Ligand Binding Assays in Non-GPCR Targets

The transition from GPCR targets to intracellular enzymes or chaperones requires high-resolution analytical platforms. While the ELISA ligand-binding assay method with antibodies may be suitable for certain biomarkers, the discovery of small-molecule inhibitors or degraders often relies on homogeneous, ratiometric readouts.

1. Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET): This technique is particularly effective for non-GPCR targets because it reduces background interference and is highly sensitive to the proximity of labeled components, making it a staple for studying protein-protein interactions.
2. Fluorescence Polarization (FP): FP is a versatile tool for high-throughput screening of E3 ligase ligands. It measures the change in the rotation of a fluorescently labeled molecule upon binding to a larger protein.
3. High-Content Screening (HCS): HCS provides spatial resolution, allowing researchers to observe exactly where a ligand binds within a living cell. This is invaluable for targets like sigma receptors, where localization is key to biological function.
4. Surface Plasmon Resonance (SPR): This label-free method is essential for moving beyond simple occupancy to understand the “on” and “off” rates of a compound, providing a deeper kinetic profile than a standard endpoint ligand binding assay.

Utilizing these receptor ligand-binding assay technologies and applications ensures that the data generated is reproducible and biophysically sound.

Practical Applications and Case Studies in Drug Discovery

Recent case studies illustrate how a well-designed ligand binding assay supports innovation beyond classical receptor pharmacology.

• Fluorescent sigma ligands for binding validation (Abatematteo FS et al. 2023)

High-affinity fluorescent sigma ligands have enabled radioligand-free displacement assays, flow cytometry, and live-cell imaging.

Nanomolar K_i values confirmed target engagement, while fluorescence-based formats expanded receptor-ligand binding assays technologies and applications to include spatial and multiparametric readouts. These tools strengthen equilibrium-based protein ligand binding assay interpretation in non-GPCR systems.

Figure 2. Confocal microscopy confirms selective σ2 receptor engagement by fluorescent ligands. Scale bar of 20 μm. Source: Abatematteo FS, et al. Development of Fluorescent 4-[4-(3H-Spiro[isobenzofuran-1,4′-piperidin]-1′-yl)butyl]indolyl Derivatives as High-Affinity Probes to Enable the Study of σ Receptors via Fluorescence-Based Techniques. J Med Chem. 2023 Mar 23;66(6):3798-3817.

• Sigma-2 receptor antagonism in Alzheimer’s disease (Rishton GM et al. 2021)

The discovery of CT1812 demonstrated that compounds identified in a phenotypic neuronal assay could displace Aβ oligomers and restore synaptic function. Subsequent profiling showed high-affinity binding to the sigma-2 receptor complex, with affinity correlating to functional rescue and cognitive improvement in vivo.

This work highlights the importance of combining functional screening with quantitative receptor ligand binding assay validation to confirm mechanism and selectivity.

• DCAF1 as an alternative E3 Ligase (Schröder M et al. 2024)

A recent study shows that recruiting DCAF1, an essential E3 ubiquitin ligase, can overcome resistance to Cereblon (CRBN)-based degraders. This is particularly important as most clinical degraders rely on a limited number of ligases, creating vulnerability to resistance mechanisms.

High-affinity DCAF1 binders were validated using SPR and TR-FRET protein ligand binding assay formats, confirming ternary complex formation under equilibrium conditions. DCAF1-based degraders successfully induced Bruton’s Tyrosine Kinase (BTK) degradation, even in CRBN-resistant models.

This case highlights the strategic value of expanding beyond traditional GPCR drug targets and reinforces the need for rigorous ligand binding assays validation and interpretation when developing next-generation degradation therapies.

At Celtarys, our mission is to expand the druggable genome by transforming targets once considered undruggable into promising candidates for clinical development. We achieve this through our proprietary chemical conjugation technology, which enables the design and delivery of high-precision fluorescent ligands tailored to specific therapeutic targets, such as sigma receptors. We also develop fluorescent probes to study protein degrader drugs like PROTACs. By bridging the gap between theoretical models and experimental validation, we’re helping researchers advance their most ambitious candidates with greater confidence.

Ready to accelerate your drug discovery program? Contact us!

References

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Hauser AS, Attwood MM, Rask-Andersen M, Schiöth HB, Gloriam DE. Trends in GPCR drug discovery: new agents, targets and indications. Nat Rev Drug Discov. 2017 Dec;16(12):829-842. doi: 10.1038/nrd.2017.178

Liu Y, Yang J, Wang T, Luo M, Chen Y, Chen C, Ronai Z, Zhou Y, Ruppin E, Han L. Expanding PROTACtable genome universe of E3 ligases. Nat Commun. 2023 Oct 16;14(1):6509. doi: 10.1038/s41467-023-42233-2

Lorente JS, Sokolov AV, Ferguson G, Schiöth HB, Hauser AS, Gloriam DE. GPCR drug discovery: new agents, targets and indications. Nat Rev Drug Discov. 2025 Jun;24(6):458-479. doi: 10.1038/s41573-025-01139-y

Rishton GM, Look GC, Ni ZJ, Zhang J, Wang Y, Huang Y, Wu X, Izzo NJ, LaBarbera KM, Limegrover CS, Rehak C, Yurko R, Catalano SM. Discovery of Investigational Drug CT1812, an Antagonist of the Sigma-2 Receptor Complex for Alzheimer’s Disease. ACS Med Chem Lett. 2021 Aug 9;12(9):1389-1395. doi: 10.1021/acsmedchemlett.1c00048

Schröder M, Renatus M, Liang X, Meili F, Zoller T, Ferrand S, Gauter F, Li X, Sigoillot F, Gleim S, Stachyra TM, Thomas JR, Begue D, Khoshouei M, Lefeuvre P, Andraos-Rey R, Chung B, Ma R, Pinch B, Hofmann A, Schirle M, Schmiedeberg N, Imbach P, Gorses D, Calkins K, Bauer-Probst B, Maschlej M, Niederst M, Maher R, Henault M, Alford J, Ahrne E, Tordella L, Hollingworth G, Thomä NH, Vulpetti A, Radimerski T, Holzer P, Carbonneau S, Thoma CR. DCAF1-based PROTACs with activity against clinically validated targets overcoming intrinsic- and acquired-degrader resistance. Nat Commun. 2024 Jan 4;15(1):275. doi: 10.1038/s41467-023-44237-4