Targeting the latent human cytomegalovirus reservoir for T-cell-mediated killing with virus-specific nanobodies.

Funder: Department of HealthLatent human cytomegalovirus (HCMV) infection is characterized by limited gene expression, making latent HCMV infections refractory to current treatments targeting viral replication. However, reactivation of latent HCMV in immunosuppressed solid organ and stem cell transplant patients often results in morbidity. Here, we report the killing of latently infected cells via a virus-specific nanobody (VUN100bv) that partially inhibits signaling of the viral receptor US28. VUN100bv reactivates immediate early gene expression in latently infected cells without inducing virus production. This allows recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals, which could serve as a therapy to reduce the HCMV latent reservoir of transplant patients

Fluorescently tagged nanobodies and NanoBRET to study ligand-binding and agonist-induced conformational changes of full-length EGFR expressed in living cells

Introduction: The Epidermal Growth Factor Receptor is a member of the Erb receptor tyrosine kinase family. It binds several ligands including EGF, betacellulin (BTC) and TGF-α, controls cellular proliferation and invasion and is overexpressed in various cancer types. Nanobodies (VHHs) are the antigen binding fragments of heavy chain only camelid antibodies. In this paper we used NanoBRET to compare the binding characteristics of fluorescent EGF or two distinct fluorescently labelled EGFR directed nanobodies (Q44c and Q86c) to full length EGFR. Methods: Living HEK293T cells were stably transfected with N terminal NLuc tagged EGFR. NanoBRET saturation, displacement or kinetics experiments were then performed using fluorescently labelled EGF ligands (EGF-AF488 or EGF-AF647) or fluorescently labelled EGFR targeting nanobodies (Q44c-HL488 and Q86c-HL488). Results: These data revealed that the EGFR nanobody Q44c was able to inhibit EGF binding to full length EGFR, while Q86c was able to recognise agonist bound EGFR and act as a conformational sensor. The specific binding of fluorescent Q44c-HL488 and EGF-AF488 was inhibited by a range of EGFR ligands (EGF> BTC>TGF-α). Discussion: EGFR targeting nanobodies are powerful tools for studying the role of the EGFR in health and disease and allow real time quantification of ligand binding and distinct ligand induced conformational changes

Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists

Funding: This research was funded by European Union’s Horizon2020 Marie Skłodowska-Curie Actions (MSCA) Program under Grant Agreement 641833 (ONCORNET) and European Cooperation in Science and Technology (COST) Action CA18133 European Research Network on Signal Transduction (ERNEST). A. Inoue was funded by the Leading Advanced Projects for Medical Innovation (LEAP) JP19gm0010004 from the Japan Agency for Medical Research and Development.Although class A G protein−coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer−dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.Publisher PDFPeer reviewe

Class III antiarrhythmic drugs amiodarone and dronedarone impair KIR2.1 backward trafficking

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. The subcellular level at which drugs interfere in trafficking pathways is largely unknown. KIR2.1 inward rectifier channels, largely responsible for the cardiac inward rectifier current (IK1), are degraded in lysosomes. Amiodarone and dronedarone are class III antiarrhythmics. Chronic use of amiodarone, and to a lesser extent dronedarone, causes serious adverse effects to several organs and tissue types, including the heart. Both drugs have been described to interfere in the late-endosome/lysosome system. Here we defined the potential interference in KIR2.1 backward trafficking by amiodarone and dronedarone. Both drugs inhibited IK1 in isolated rabbit ventricular cardiomyocytes at supraclinical doses only. In HK-KWGF cells, both drugs dose- and time-dependently increased KIR2.1 expression (2.0 ± 0.2-fold with amiodarone: 10 μM, 24 hrs; 2.3 ± 0.3-fold with dronedarone: 5 μM, 24 hrs) and late-endosomal/lysosomal KIR2.1 accumulation. Increased KIR2.1 expression level was also observed in the presence of Nav1.5 co-expression. Augmented KIR2.1 protein levels and intracellular accumulation were also observed in COS-7, END-2, MES-1 and EPI-7 cells. Both drugs had no effect on Kv11.1 ion channel protein expression levels. Finally, amiodarone (73.3 ± 10.3% P < 0.05 at −120 mV, 5 μM) enhanced IKIR2.1 upon 24-hrs treatment, whereas dronedarone tended to increase IKIR2.1 and it did not reach significance (43.8 ± 5.5%, P = 0.26 at −120 mV; 2 μM). We conclude that chronic amiodarone, and potentially also dronedarone, treatment can result in enhanced IK1 by inhibiting KIR2.1 degradation

Class III antiarrhythmic drugs amiodarone and dronedarone impair KIR2.1 backward trafficking

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. The subcellular level at which drugs interfere in trafficking pathways is largely unknown. KIR2.1 inward rectifier channels, largely responsible for the cardiac inward rectifier current (IK 1), are degraded in lysosomes. Amiodarone and dronedarone are class III antiarrhythmics. Chronic use of amiodarone, and to a lesser extent dronedarone, causes serious adverse effects to several organs and tissue types, including the heart. Both drugs have been described to interfere in the late-endosome/lysosome system. Here we defined the potential interference in KIR2.1 backward trafficking by amiodarone and dronedarone. Both drugs inhibited IK 1 in isolated rabbit ventricular cardiomyocytes at supraclinical doses only. In HK-KWGF cells, both drugs dose- and time-dependently increased KIR2.1 expression (2.0 ± 0.2-fold with amiodarone: 10 μM, 24 hrs; 2.3 ± 0.3-fold with dronedarone: 5 μM, 24 hrs) and late-endosomal/lysosomal KIR2.1 accumulation. Increased KIR2.1 expression level was also observed in the presence of Nav1.5 co-expression. Augmented KIR2.1 protein levels and intracellular accumulation were also observed in COS-7, END-2, MES-1 and EPI-7 cells. Both drugs had no effect on Kv11.1 ion channel protein expression levels. Finally, amiodarone (73.3 ± 10.3% P < 0.05 at −120 mV, 5 μM) enhanced IKIR 2.1 upon 24-hrs treatment, whereas dronedarone tended to increase IKIR 2.1 and it did not reach significance (43.8 ± 5.5%, P = 0.26 at −120 mV; 2 μM). We conclude that chronic amiodarone, and potentially also dronedarone, treatment can result in enhanced IK 1 by inhibiting KIR2.1 degradation

The human cytomegalovirus-encoded G protein- coupled receptor UL33 exhibits oncomodulatory properties

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein- coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gαq, Gαi, and Gαs proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies

Indole-3-carbinol-induced modulation of NF-kB signalling is breast cancer cell-specific and does not correlate with cell death

Indole-3-carbinol (I3C), a dietary chemopreventive compound, induces cell death in human breast cancer cells by modulating activities of Src and epidermal growth factor receptor (EGFR). The effect of I3C on NF-κB, constitutively activated in breast cancer cells, was investigated. Nuclear extracts of MDA-MB-468, MDA-MB-231 and HBL100 cells contained all of the Rel proteins with similar expression patterns in the latter two. The level of NF-κB-regulated reporter gene expression was in the order HBL100 << MDA-MB-468 << MDA-MB-231. Upstream inhibition, using PI3K, EGFR or IKKβ inhibitors, resulted in cell-specific effects on expression of the NF-κB-regulated reporter gene and endogenous genes Bcl-xL, IκBα and IL-6, as well as on cell viability. The expression patterns of Rel and several NF-κB-regulated genes and the response to LY249002 in MDA-MB-468 cells contrasted with those in other cells. I3C induced NF-κB-regulated reporter gene expression at 12 h in MDA-MB-468 cells. Conversely, it was reduced at 24 h in HBL100 cells. I3C treatment for 6 h alone or in combination with TNFα induced NF-κB-regulated reporter gene expression, detected 5 h later, in MDA-MB-468, but not HBL100 cells. I3C induced NF-κB p65/p50 DNA binding at 6.5 h, preceded by association of IKKβ with the Src/EGFR complex and increased phospho-IκBα in MDA-MB468 cells. TNFα increased I3C-induced apoptosis in MDA-MB-468 and MDA-MB-231 cells. It also induced apoptosis, enhanced by I3C, in HBL100 cells. Hence, regulation of constitutive NF-κB was cell-specific. I3C influenced the NF-κB pathway in a cell-specific manner, which was not related to apoptosis. However, the combination of I3C and TNFα increased apoptosis in all cell lines

Nanobodies detecting and modulating GPCRs outside in and inside out

G protein-coupled receptors (GPCRs), belonging to the largest class of membrane proteins, play a prominent role in many (patho)physiological processes and are, therefore, important drug targets. Although most often targeted by small molecules, these receptors have become interesting targets for antibodies and antibody fragments, especially camelid-derived heavy chain-only antibodies and fragments thereof (nanobodies). The small size and molecular structure of nanobodies allow GPCR-binding and modulation, from both the intracellular and extracellular sides. These molecular features make nanobodies attractive tools to study, modulate, and exploit GPCRs. Besides modulating GPCR activity as monovalent or multivalent constructs, nanobodies can also be functionalized for imaging and therapy. Moreover, GPCR-binding nanobodies have been instrumental in obtaining crystal structures of GPCRs, facilitating structure-based drug discovery. Here, we describe the current status and future perspectives of nanobodies targeting GPCRs intra and extracellularly