The NanoBiT-Based Homogenous Ligand–Receptor Binding Assay

Liu, Yali; Guo, Zhan‐Yun

doi:10.1007/978-1-0716-2473-9_10

Cited by 4 publications

(5 citation statements)

References 25 publications

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“…Nonetheless, the small peptide HiBiT is crucial for the efficient assembly of Vpr into virions. The HiBiT peptide needs to fit into the binding pocket of LgBiT for detection of NLuc activity [55]; therefore, compounds, such as DrkBiT [56], interfering with the binding between HiBiT and LgBiT may be selected as false candidates. Hence, to avoid the selection of incorrect candidates, direct quantification, which measures p24 Gag in the cell supernatant, is essential for determining candidate compounds by secondary screening.…”

Section: Discussionmentioning

confidence: 99%

A heterocyclic compound inhibits viral release by inducing cell surface BST2/Tetherin/CD317/HM1.24

Nyame,

Togami,

Yoshida

et al. 2024

Preprint

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The introduction of combined antiretroviral therapy (cART) has greatly improved the quality of life of human immunodeficiency virus type 1 (HIV-1)-infected individuals. Nonetheless, the ever-present desire to seek out a full remedy for HIV-1 infections makes the discovery of novel antiviral medication compelling. Owing to this, a new late-stage inhibitor, Lenacapavir/Sunlenca, an HIV multi-phase suppressor, was clinically authorized in 2022. Besides unveiling cutting-edge antivirals inhibiting late-stage proteins or processes, newer therapeutics targeting host restriction factors hold promise for the curative care of HIV-1 infections. Notwithstanding, bone marrow stromal antigen 2 (BST2)/Tetherin/CD317/HM1.24, which entraps progeny virions is an appealing HIV-1 therapeutic candidate. In this study, a novel drug screening system was established, using the Jurkat/Vpr-HiBiT T cells, to identify drugs that could obstruct HIV-1 release; the candidate compounds were selected from the Ono Pharmaceutical compound library. Jurkat T cells expressing Vpr-HiBiT were infected with NL4-3, and the amount of virus release was quantified indirectly by the amount of Vpr-HiBiT incorporated into the progeny virions. Subsequently, the candidate compounds that suppressed viral release were used to synthesize the heterocyclic compound, HT-7, which reduces HIV-1 release with less cellular toxicity. Notably, HT-7 increased cell surface BST2 coupled with HIV-1 release reduction in Jurkat cells but not Jurkat/KO-BST2 cells. Seemingly, HT-7 impeded simian immunodeficiency virus (SIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) release. Concisely, these results suggest that the reduction in viral release, following HT-7 treatment, resulted from the modulation of cell surface expression of BST2 by HT-7.

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Section: Discussionmentioning

confidence: 99%

A heterocyclic compound inhibits viral release by inducing cell surface BST2/Tetherin/CD317/HM1.24

Nyame,

Togami,

Yoshida

et al. 2024

Preprint

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“…To test whether mature FAM237A is a ligand of GPR83, we first employed the NanoBiT-based ligandreceptor binding assay that was recently developed by our laboratory and has been validated using certain GPCRs, such as relaxin family peptide receptor (RXFP) 3 and 4, and growth hormone secretagogue receptor type 1a (GHSR1a) [18][19][20]. To set up this binding assay, an inactive secretory large NanoLuc fragment for Nano-BiT (sLgBiT) was genetically fused to the extracellular N-terminus of the mature human GPR83 (Fig.…”

Section: Mature Fam237a Binds To Gpr83 With High Affinity and High Sp...mentioning

confidence: 99%

FAM237A, rather than peptide PEN and proCCK56‐63, binds to and activates the orphan receptor GPR83

Wang

Shao

et al. 2023

The FEBS Journal

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“…To determine if PRC1-Hinge-Fc competed with FSH binding on FSHR, a luminescence assay based on a split luciferase fused to proteins of interest was conducted [42]. FSH was fused to the LgBit subunit of the luciferase and produced in ExpiCHO cells.…”

Section: Nanobit Fsh Binding Assaymentioning

confidence: 99%

Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHR

Zehnaker,

Vallet,

Gourdon

et al. 2023

IJMS

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Developing modulatory antibodies against G protein-coupled receptors is challenging. In this study, we targeted the follicle-stimulating hormone receptor (FSHR), a significant regulator of reproduction, with variable domains of heavy chain-only antibodies (VHHs). We built two immune VHH libraries and submitted them to multiplexed phage display approaches. We used next-generation sequencing to identify 34 clusters of specifically enriched sequences that were functionally assessed in a primary screen based on a cAMP response element (CRE)-dependent reporter gene assay. In this assay, 23 VHHs displayed negative or positive modulation of FSH-induced responses, suggesting a high success rate of the multiplexed strategy. We then focused on the largest cluster identified (i.e., PRC1) that displayed positive modulation of FSH action. We demonstrated that PRC1 specifically binds to the human FSHR and human FSHR/FSH complex while potentiating FSH-induced cAMP production and Gs recruitment. We conclude that the improved selection strategy reported here is effective for rapidly identifying functionally active VHHs and could be adapted to target other challenging membrane receptors. This study also led to the identification of PRC1, the first potential positive modulator VHH reported for the human FSHR.

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The NanoBiT-Based Homogenous Ligand–Receptor Binding Assay

Cited by 4 publications

References 25 publications

A heterocyclic compound inhibits viral release by inducing cell surface BST2/Tetherin/CD317/HM1.24

A heterocyclic compound inhibits viral release by inducing cell surface BST2/Tetherin/CD317/HM1.24

FAM237A, rather than peptide PEN and proCCK56‐63, binds to and activates the orphan receptor GPR83

Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHR

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