Quantification of Bioorthogonal Stability in Immune Phagocytes Using Flow Cytometry Reveals Rapid Degradation of Strained Alkynes

Bakkum, Thomas; Leeuwen, Tyrza van; Sarris, Alexi J C; Elsland, Daphne M. van; Poulcharidis, Dimitrios; Overkleeft, Herman S.; Kasteren, Sander I. van

doi:10.1021/acschembio.8b00355

Cited by 17 publications

(20 citation statements)

References 76 publications

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“…Hpg modification did not affect BM‐DC uptake (Figure S4).The rate of DsRed expression after uptake in both Met‐ and Hpg‐grown strains was equal, indicating no detectable impact of Hpg on intracellular survival or proliferation (Figures C and S4). Furthermore, bioorthogonal ligation within BM‐DCs showed no reduction in click signal, confirming our previous finding that nonstrained alkynes were stable in the phagolysosomal pathway (Figures D and S4).…”

Section: Resultssupporting

confidence: 88%

Ultrastructural Imaging of Salmonella–Host Interactions Using Super‐resolution Correlative Light‐Electron Microscopy of Bioorthogonal Pathogens

et al. 2018

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The imaging of intracellular pathogens inside host cells is complicated by the low resolution and sensitivity of fluorescence microscopy and by the lack of ultrastructural information to visualize the pathogens. Herein, we present a new method to visualize these pathogens during infection that circumvents these problems: by using a metabolic hijacking approach to bioorthogonally label the intracellular pathogen Salmonella Typhimurium and by using these bioorthogonal groups to introduce fluorophores compatible with stochastic optical reconstruction microscopy (STORM) and placing this in a correlative light electron microscopy (CLEM) workflow, the pathogen can be imaged within its host cell context Typhimurium with a resolution of 20 nm. This STORM‐CLEM approach thus presents a new approach to understand these pathogens during infection.

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Section: Resultssupporting

confidence: 88%

Ultrastructural Imaging of Salmonella–Host Interactions Using Super‐resolution Correlative Light‐Electron Microscopy of Bioorthogonal Pathogens

et al. 2018

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“…This alkyne moiety can be conjugated to CalFluor-488, which increases the fluorescence signal by two orders of magnitude, resulting in a low signal-to-noise ratio (Shieh et al., 2015). Moreover, the alkyne moiety is only three carbon atoms in size and does not (or less) perturb the molecular mechanisms of antigen processing and presentation, in contrast to large fluorophores, and survives the harsh environment in lysosomes (Bakkum et al., 2018). By incubating human dendritic cells with bio-orthogonal functionalized peptide HA 318-338 , we could show increased MHC class II presentation over time for at least up to 5-hr incubation (Figures 6B–6D and S8).…”

Section: Resultsmentioning

confidence: 99%

The Phosphoinositide Kinase PIKfyve Promotes Cathepsin-S-Mediated Major Histocompatibility Complex Class II Antigen Presentation

et al. 2019

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SummaryAntigen presentation to T cells in major histocompatibility complex class II (MHC class II) requires the conversion of early endo/phagosomes into lysosomes by a process called maturation. Maturation is driven by the phosphoinositide kinase PIKfyve. Blocking PIKfyve activity by small molecule inhibitors caused a delay in the conversion of phagosomes into lysosomes and in phagosomal acidification, whereas production of reactive oxygen species (ROS) increased. Elevated ROS resulted in reduced activity of cathepsin S and B, but not X, causing a proteolytic defect of MHC class II chaperone invariant chain Ii processing. We developed a novel universal MHC class II presentation assay based on a bio-orthogonal “clickable” antigen and showed that MHC class II presentation was disrupted by the inhibition of PIKfyve, which in turn resulted in reduced activation of CD4+ T cells. Our results demonstrate a key role of PIKfyve in the processing and presentation of antigens, which should be taken into consideration when targeting PIKfyve in autoimmune disease and cancer.

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“…Fluorescence has a broad application as a modality for in vitro and in vivo imaging. 1,2 In the clinical field, an increasing interest in fluorescence imaging has been triggered by the introduction of fluorescence camera systems that can be applied during surgery. 3−5 In this context, real-time fluorescence imaging is utilized to visualize a distinct (e.g., diseased) tissue type within the surgical field.…”

Section: Introductionmentioning

confidence: 99%

Click Chemistry in the Design and Production of Hybrid Tracers

et al. 2019

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Hybrid tracers containing both fluorescent and radioactive imaging labels have demonstrated clinical potential during sentinel lymph node procedures. To combine these two labels on a single targeting vector that allows tumor-targeted imaging, end-labeling strategies are often applied. For α v β 3 -integrin-targeting hybrid tracers, providing an excellent model for evaluating tracer development strategies, end-labeling-based synthesis provides a rather cumbersome synthesis strategy. Hence, the aim of this study was to investigate the use of heterobifunctional cyanine dyes in a click-chemistry-based synthesis strategy for RGD-based hybrid tracers. The triazole-based hybrid tracers DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] were obtained in fewer steps than DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK] and had partition coefficients of log P (o/w) = −2.55 ± 0.10, −1.45 ± 0.03, and −2.67 ± 0.12, respectively. Both tracers were chemically stable, and the brightnesses of DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] were, respectively, 23 × 10 3 and 40 × 10 3 M –1 cm –1 ; lower than that of the reference tracer DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK] (50 × 10 3 M –1 cm –1 ). Assessment of serum protein binding revealed no statistically significant difference (44 ± 2 and 40 ± 2% bound for DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] , respectively; 36 ± 5% bound for DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK] ; p > 0.05). DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] ( K D = 17.5 ± 6.0) had a statistically significantly higher affinity than the reference compound DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK] ( K D = 30.3 ± 5.7; p < 0.0001), but DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] had a statistically significantly lower affinity ( K...

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Quantification of Bioorthogonal Stability in Immune Phagocytes Using Flow Cytometry Reveals Rapid Degradation of Strained Alkynes

Cited by 17 publications

References 76 publications

Ultrastructural Imaging of Salmonella–Host Interactions Using Super‐resolution Correlative Light‐Electron Microscopy of Bioorthogonal Pathogens

Ultrastructural Imaging of Salmonella–Host Interactions Using Super‐resolution Correlative Light‐Electron Microscopy of Bioorthogonal Pathogens

The Phosphoinositide Kinase PIKfyve Promotes Cathepsin-S-Mediated Major Histocompatibility Complex Class II Antigen Presentation

Click Chemistry in the Design and Production of Hybrid Tracers

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