Kaposi's sarcoma-associated herpesvirus (KSHV) is a common virus with severe outcome and no effective antiviral treatment. KSHV encodes the constitutive active chemokine receptor ORF74 with broad-spectrum CXC-chemokine binding. Here, we leverage ORF74's mimic of endogenous receptors to design chemokine-based immunotoxins for selective killing of KSHV-infected cells.
Four CXC-chemokines with high affinity to ORF74 were fused to domain II, IB, and III of Pseudomonas exotoxin A to generate fusion toxin proteins (FTPs). FTP-induced cell killing was tested in cells expressing ORF74 or one of four chemokine receptors (CXCR1-4). Internalization of all receptors was probed using SNAP-tagged receptors. Second-generation FTPs were designed from receptor structures and molecular modelling to increase selectivity for ORF74 over CXCR1-4. Finally, antiviral activity of FTPs was tested using genetically engineered KSHV.
FTPs, based on the agonists (CXCL1, and -8) and inverse agonists (CXCL10 and -12) of ORF74 potently killed ORF74-expressing cells. The inverse agonist based FTPs leveraged constitutive internalization for efficient toxin delivery via ORF74, whereas agonists increased internalization further. CXCL10-FTP had the strongest cell-killing and, as the only FTP, selectivity for ORF74 over its endogenous receptor, CXCR3. Second-generation FTPs improved this selectivity from 25-fold to 126-fold by the mutation (R8D) in CXCL10-FTP, designed to lose ionic interaction within CXCR3's main binding pocket. Both inverse agonist-based FTPs effectively prevented KSHV-reactivation.
Our findings highlight the versatility of FTPs in precise delivery of toxin payloads and provide a foundation for potential applications in antiviral and anticancer therapies targeting KSHV-associated diseases.
Copyright © 2026 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

The glucagon-like peptide 1 receptor (GLP1R) is a major drug target with several agonists being prescribed in individuals with type 2 diabetes and obesity1,2. The impact of genetic variability of GLP1R on receptor function and its association with metabolic traits are unclear with conflicting reports. Here, we show an unexpected diversity of phenotypes ranging from defective cell surface expression to complete or pathway-specific gain of function (GoF) and loss of function (LoF), after performing a functional profiling of 60 GLP1R variants across four signalling pathways. The defective insulin secretion of GLP1R LoF variants is rescued by allosteric GLP1R ligands or high concentrations of exendin-4/semaglutide in INS-1 823/3 cells. Genetic association studies in 200,000 participants from the UK Biobank show that impaired GLP1R cell surface expression contributes to poor glucose control and increased adiposity with increased glycated haemoglobin A1c and body mass index. This study defines impaired GLP1R cell surface expression as a risk factor for traits associated with type 2 diabetes and obesity and provides potential treatment options for GLP1R LoF variant carriers.
© 2023. The Author(s), under exclusive licence to Springer Nature Limited.

The viral G-protein-coupled receptor (vGPCR) BILF1 encoded by the Epstein-Barr virus (EBV) is an oncogene and immunoevasin and can downregulate MHC-I molecules at the surface of infected cells. MHC-I downregulation, which presumably occurs through co-internalization with EBV-BILF1, is preserved among BILF1 receptors, including the three BILF1 orthologs encoded by porcine lymphotropic herpesviruses (PLHV BILFs). This study aimed to understand the detailed mechanisms of BILF1 receptor constitutive internalization, to explore the translational potential of PLHV BILFs compared with EBV-BILF1.
A novel real-time fluorescence resonance energy transfer (FRET)-based internalization assay combined with dominant-negative variants of dynamin-1 (Dyn K44A) and the chemical clathrin inhibitor Pitstop2 in HEK-293A cells was used to study the effect of specific endocytic proteins on BILF1 internalization. Bioluminescence resonance energy transfer (BRET)-saturation analysis was used to study BILF1 receptor interaction with β-arrestin2 and Rab7. In addition, a bioinformatics approach informational spectrum method (ISM) was used to investigate the interaction affinity of BILF1 receptors with β-arrestin2, AP-2, and caveolin-1.
We identified dynamin-dependent, clathrin-mediated constitutive endocytosis for all BILF1 receptors. The observed interaction affinity between BILF1 receptors and caveolin-1 and the decreased internalization in the presence of a dominant-negative variant of caveolin-1 (Cav S80E) indicated the involvement of caveolin-1 in BILF1 trafficking. Furthermore, after BILF1 internalization from the plasma membrane, both the recycling and degradation pathways are proposed for BILF1 receptors.
The similarity in the internalization mechanisms observed for EBV-BILF1 and PLHV1-2 BILF1 provide a foundation for further studies exploring a possible translational potential for PLHVs, as proposed previously, and provides new information about receptor trafficking.
© 2023. The Author(s).

Inhibiting receptor tyrosine kinases is commonly achieved by two main strategies targeting either the intracellular kinase domain by low molecular weight compounds or the extracellular ligand-binding domain by monoclonal antibodies. Identifying small molecules able to inhibit RTKs at the extracellular level would be highly desirable to gain exquisite selectivity but is believed to be challenging owing to the size of RTK endogenous ligands (cytokines, growth factors) and the topology of RTK extracellular domains. We here report the high-throughput screening of the French Chemical Library (48K compounds) for extracellular inhibitors of the Fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase, by a homogeneous time-resolved fluorescence competition assay. A total of 679 small molecular weight ligands (1.4%) were confirmed to strongly inhibit (>75%) the binding of the fluorescent labeled FLT3 ligand (FL cytokine) to FLT3 overexpressed in HEK-293 cells, at two different concentrations (5 and 20 μM). Concentration-response curves, obtained for 111 lead-like molecules, confirmed the unexpected tolerance of the FLT3 extracellular domain for low molecular weight druggable inhibitors exhibiting submicromolar potencies, chemical diversity, and promising pharmacokinetic properties. Further investigation of one hit confirmed inhibitory properties in dorsal root ganglia neurons and in a mouse model of neuropathic pain.

The SARS-CoV-2 coronavirus infects human cells through the interaction of the viral envelope spike protein (IPR044366) with the human angiotensin-converting enzyme 2 (ACE2), expressed at the surface of target cells. Here, we describe a detailed protocol to measure the binding of the receptor binding domain (RBD) of spike to ACE2 by time-resolved fluorescence resonance energy transfer (TR-FRET). The assay detects the spike/ACE2 interaction in physiologically relevant cellular contexts and is suitable for high-throughput investigation of interfering small-molecule compounds and antibodies. For complete details on the use and execution of this protocol, please refer to Cecon et al. (2021).
© 2021 The Author(s).