Biomolecular interaction is a universal component and of key importance to most biological processes, which plays a pivotal role in pharmacology. In recent years, the landscape of biomolecular interaction significantly expanded due to advances in a number of ingenious biochemical and biophysical technologies. Bioluminescence resonance energy transfer (BRET) is a naturally occurred physical phenomenon, and has been engineered to monitor direct molecular interactions, due to its stringent distance requirement (≤ 10 nm) to allow efficient energy transfer. However, the weak signal and broad spectrum of conventional BRET donor significantly hindered its application. Recently, a surge of the development of novel luciferase, NanoLuc®, as BRET donor brings a new wave of exploring various molecular interactions. Herein, we provide a review of the recent advances of NanoLuc-based BRET technologies in studying biomolecular interactions, particularly in pharmacological applications.

Keywords: Bioluminescence Resonance Energy Transfer; Nanoluc Luciferase; Protein-Protein Interaction; Drug-Target Interaction; Intramolecular BRET Biosensor.

Figure 1. Schematic illustration of robust BRETn platform.

Stoddart et al. {C}[11]{C}{C} for the first time developed a series of intramolecular NanoBRET biosensors and demonstrated their potential application in monitoring drug-protein interactions, particularly using the ligand binding to G-protein-coupled receptors (GPCRs) as an example. These NanoBRET biosensors are consisted by fluorophore-labeled ligand and NLuc-tagged proteins. They used these NanoBRET biosensors to characterize the binding affinity of a panel of ligand to their specific GPCRs, such as -adrenergic receptor, adenosine receptor and angiotensin receptors, in both saturation and competitive binding assays in live cells without any additional washing and lysing steps. Similar specificity and parameters were obtained from these example studies compared with the data from previous conventional methods, suggesting NanoBRET a reliable technology for studying drug-protein interactions [12].

Robers et al. {C}[13]{C} utilized similar strategy and further extended this method to study other protein target classes, such as histone deacetylase and bromodomain-containing proteins. Noteworthy, similar as radio-labeled ligands, careful selection of labeling fluorophore is needed to minimize the labeling-dependent affinity shift.

5. Intramolecular NLuc-based BRET biosensors reveal protein dynamics

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In contrast with the above mentioned BRET biosensors that enabling the study of intermolecular interactions, the intramolecular BRET biosensor configuration, by leveraging the protein conformational change between BRET on and off status upon protein in/activation, provides a unique avenue to study protein dynamics at various conditions.

Recently, Wang et al. {C}[14,15]{C}{C} used the principle of NanoBRET technology to decipher the mechanism of ERK1/2 phosphorylation of Rabin8, a guanine nucleotide exchange factor that regulates exocytosis. In order to test the hypothesis that Rabin8 adopts an autoinhibitory conformation upon ERK1/2 phosphorylation, Wang et al. constructed a Rabin8 intramolecular BRET sensor by tagging NLuc and HaloTag at its N- and C-terminal, respectively. In their hypothesized model, Rabin8 should undergo conformational change between closed inhibitory dephosphorylated conformation with low BRET signal and open active phosphorylated conformation with high BRET signal. Indeed, Wang et al. successfully observed that Rabin8 BRET sensor with kinase dead ERK2 had a significantly higher BRET signal than that with constitutively active ERK2, and consequently confirmed their hypothesized model that Rabin8 can inhibit its own catalytic activity by having the C-terminus block the guanine nucleotide exchange factor domain activity.

Besides monitoring protein dynamics, intramolecular BRET biosensor is also powerful to study drug-protein interactions. Johnsson et al. [16] for the first time invented luciferased-based indicators of drugs (LUCIDs). The key component underlying LUCIDs is an engineered ligand-Cy3-SNAP-NLuc-protein (LiCSNP) fusion construct. This LiCSNP construct can undergo conformational change from closed high BRET to open low BRET conformation upon competitive binding of external ligand with the intramolecular ligand to the receptor protein. By altering the proteins of interest (including dihydrofolate reductase, FKBP12, cyclophilin A, carbonic anhydrase II and DIG10.3), Johnsson et al. have demonstrated the validity of using engineered LUCIDs to monitor diverse drugs (including immunosuppressants, antiepileptics, anticancer and antirrhythmics agents) in patient samples with portable devices. By introducing a secondary tethered ligand in LUCIDs setup, Johnsson et al. [17] further developed a chemical ligand-associated steric hindrance (CLASH) approach to modulate protein activity using unrelated effectors.

In 2015, NLuc-based BRET technologies have transformed the study of molecular interactions from diverse aspects, including protein-protein interaction, drug-protein interaction and engineered protein biosensors. This success is largely due to the advantages of the superior optical and biochemical properties of NLuc which enables the robust detection of molecular interactions in a physiological relevant context. The increased demand and expanded landscape of molecular interaction networks would encourage exploitation of the current and future potential of NLuc-based BRET technologies.

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