The identification of novel therapeutics through target-based drug discovery requires precise quantification of cellular and biochemical activities, which are essential for elucidating the mechanistic functions of biomolecules and their dysregulation in pathological conditions. Such quantitative analyses facilitate the development of therapeutic interventions aimed at modulating specific molecular processes to prevent or treat diseases. Fluorescence- and luminescence-based assays are widely utilized for their accuracy and robustness in measuring the in vitro activity of molecular targets, as well as for identifying novel chemical entities capable of modulating target function. Various types of fluorescence- and luminescence-based technologies such as fluorescence polarization (FP), time resolved Förster resonance energy transfer (TR-FRET), Homogeneous Time-Resolved Fluorescence (HTRF), bioluminescence resonance energy transfer (BRET), NanoBRET, and NanoBiT are currently being employed in drug discovery for characterizing protein-protein interaction (PPI), ligand-receptor dynamics, intracellular signalling pathways and drug mechanism of action.

FRET and BRET assay capabilities at o2h

FRET and BRET are proximity-based target engagement assays that offer significant advantages over conventional fluorescence and luminescence assays, including a homogeneous format, ratiometric readout, low background noise, and miniaturization potential. These techniques enable rapid and sensitive detection of molecular interactions in a non-invasive manner. They are particularly useful for identifying small-molecule inhibitors that disrupt pathological protein-protein interactions (PPIs). Additionally, FRET and BRET technologies are widely employed to characterize receptor-ligand interactions by assessing ligand binding affinity, receptor activation, and real-time conformational changes, providing crucial insights for drug development. They also facilitate the study of intracellular signalling pathways, including kinase activation, secondary messenger production, and protein modifications.

o2h offers a wide range of custom-tailored FRET- and BRET-based assay capabilities to accelerate your research and development objectives, by leveraging our in-house PHERAstar FSX and the CLARIOstar Plus multimode plate readers from BMG Labtech. Our expertise includes screening PPI inhibitors and assessing the potency of GPCR modulators through HTRF-based approaches. We also evaluate kinase activity inhibitors using both recombinant proteins and cell lysates via HTRF assays. Our scientists possess extensive experience in live-cell target engagement studies using NanoBRET assays. Additionally, we employ BRET assays to characterize the kinetics of PROTAC-induced ternary complex formation between target protein and E3 ligase in live cells. To optimize efficiency and conserve reagents, we offer miniaturized FRET- and BRET-based bioassays in a 384-well format, enabling high-throughput screening.

FRET assay – Case study 1

o2h Discovery specializes in GPCR biology, offering opportunities to screen compounds that act as agonists, partial agonists, or antagonists for neuropsychiatric disorders targeting serotonin (5-HT, 5-hydroxytryptamine) receptors. Among these, the serotonin 2A receptor (5-HT2A) is particularly significant for its role in mood regulation, cognition, and perception. Activation of serotonin signaling triggers downstream events such as intracellular Ca2+ release, inositol monophosphate (IP1) formation, and β-arrestin2 recruitment to the plasma membrane.

At o2h, we have developed and screened a series of 5-HT2A-targeting small molecules using the IP1 Gq HTRF assay from Revvity. This competitive immunoassay measures IP1 accumulation in cells stably expressing the target receptor. HTRF™ (Homogeneous Time-Resolved Fluorescence) is a no-wash technology that combines standard FRET with time-resolved fluorescence measurement, effectively eliminating short-lived background signals. In this assay, native IP1 produced by the cells or unlabeled IP1 (standard curve) competes with d2-labeled IP1 (acceptor) for binding to anti-IP1-Cryptate (donor). The resulting ratio metric fluorescence readout (665 nm/620 nm) allows for precise quantification of IP1 accumulation using the IP1 standard curve.

Figure 1: (A) Intracellular IP1 levels measured using the HTRF kit in stably expressing 5-HT2A cells stimulated with increasing concentrations of serotonin (EC50 = 9.8 nM).
(B) Classification of compounds as agonists, demonstrating varying potencies and efficacies.
(C) Inhibition of IP1 generation in 5-HT2A expressing cells treated with increasing concentrations of the antagonist ketanserin, following stimulation with an EC80 concentration of serotonin (5-HT).

FRET assay – Case study 2

o2h provides a wide array of commercial and custom-tailored kinase activity and selectivity assays designed to expedite the development of kinase-targeted therapies. Leveraging advanced platforms such as high-throughput screening and both biochemical and cellular kinase assays, our team facilitates the discovery of potent and selective kinase inhibitors across all stages of drug development, from hit identification to lead optimization.

We undertook a discovery campaign against a serine/threonine-protein kinase, which is a therapeutic target in breast cancer due to its ability to prevent ubiquitin-mediated proteasomal degradation of estrogen receptor α (ERα). o2h scientists have successfully identified small molecule inhibitors against the kinase using the HTRF KinEASE STK S1 kit from Revvity as the primary screening assay. This kit provides a simple biochemical approach to study serine/threonine kinase activity, screen for inhibitors and characterize them in a 384-well format. The kinase phosphorylates the STK-S1 biotin substrate in presence of ATP. The phosphorylation is detected using STK antibody-Eu3+-Cryptate and Streptavidin-XL665. The resulting ratio metric fluorescence readout (665 nm/620 nm) allows for precise quantification of the serine/threonine kinase activity and inhibition potency of novel kinase inhibitors.

Figure 2: Tool compound mediated inhibition of WT kinase activity (IC50 = 461 nM). The HTRF ratios of the test compounds were normalized using the positive and negative assay controls to measure percentage kinase activity. % Inhibition = 100 – % Activity.

FRET assay – Case study 3

o2h familiarity with offering effective HTRF assays to support drug development spanning multiple phases can be seen in a recent project where complimentary assays were used to provide both biochemical assay and subsequent cellular assay steps. Both steps made use of Revvity HTRF assays tailored to the target biology. The primary assay detected small molecule mediated disruption of protein-protein interaction (PPI) of a recombinant target kinase and its cognate signalling binding partner in a cell-free environment. Downstream in the assay cascade, a separate HTRF assay was run in K562 cells to monitor target engagement of active compounds in cells via a target-dependent signalling cascade ending with modulation of a specific protein phosphorylation marker. Both assays were optimised for low volume 384 plates, reported IC50 values in keeping with prior characterisation for key tool compounds and identified lead molecules with evidence of activity and cell permeability.

Figure 3: Development and characterization of PPI inhibitors against a target kinase, utilizing biochemical followed by cellular screening assays, employing HTRF readout technology

BRET assay – Case study 1

o2h offers a broad repertoire of biochemical, cell-based and biophysical target engagement assays for screening of putative ligands against multiple therapeutic targets. NanoBRET technology presents an attractive and high-throughput assay format for investigating target engagement in a cellular setting. NanoBRET assay measures apparent affinity of small molecules by competitive displacement of a target specific tracer, reversibly bound to target of interest fused to a NanoLuc tag.

o2h biologists have extensive experience in establishing NanoBRET assay for different targets and have screened >1000 in-house synthesized small molecules, supporting hit to lead identification with an average turn-around-time of 3-4 days. We have presented a case study below showing NanoBRET assay development, validation and compound screening for a kinase target involved in cell cycle regulation and DNA damage repair.

Figure 4: NanoBRET assay validation and compound screening. (A) Tool compound showing a dose-dependent displacement of the tracer with the optimal inhibition at tracer concentrations at 0.5 μM and 1 μM. (B) An example illustrating inter-day and inter-person variability with tool compound CTx-0294885 performed as a 5-point DRC as indicated above. (C) Representative dose-response curves of compounds with different potencies along with the tool/control compound. (D) Time kinetics of a potent compound showing displacement of the tracer in comparison with an inactive compound.

BRET assay – Case study 2

o2h scientists have extensive experience in developing BRET-based assays for investigating the key features of PROTACs and TPD molecules like ternary complex formation, screen for endogenous protein degradation and estimate DC50 values (half maximum degradation concentration). We offer integrated PROTAC services as well as a bespoke and customisable “off-the-shelf” PROTAC toolbox to jump-start your targeted protein degradation program, along with our strong expertise in medicinal and synthetic chemistry.

As a case study, we investigated the ternary complex formation induced by a BRD4 PROTAC MZ1 using a NanoBRET® assay system from Promega, where the BRD4 (BD2) domain was tagged with Nanoluciferase and the recruited VHL E3 ligase tagged with a Halotag. Using this system, we were able to quantify the interaction that occurs between them in presence of MZ1 by measuring the level of BRET signal emitted by the Halotag which increases upon close proximity with the nano luciferase.

Figure 5: MZ1 PROTAC induced ternary complex formation (A – endpoint, B – kinetics) and BRD4 protein degradation (C – endpoint, D – kinetics) using NanoLuc-BRD4 and VHL-Halo Tag NanoBRET expression system. MZ1 induced higher BRET ratio and lower luminescence indicates stable ternary complex formation and sustained protein degradation which was rescued in the presence of the proteasome inhibitor MG132.

Together, these capabilities enhance our drug discovery efforts and contribute to a deeper understanding of complex biological systems.

To know more about our biology services offering or to request our brochure, please reach out to us at discovery@o2h.com.