Spacer-free BODIPY fluorogens in antimicrobial peptides for direct imaging of fungal infection in human tissue

Mendive‐Tapia, Lorena; Zhao, Can; Akram, Ahsan R.; Preciado, Sara; Alberício, Fernando; Lee, Martin; Serrels, Alan; Kielland, Nicola; Read, Nick D.; Lavilla, Rodolfo; Vendrell, Marc

doi:10.1038/ncomms10940

Cited by 116 publications

(99 citation statements)

References 64 publications

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“…49–51 Functionalized BODIPYs can be conjugated to peptides or proteins for imaging applications using “click” reactions, 44,52–54 traditional amide bond formation reactions, 30,55–58 or solid support methodologies. 59 However, several challenges have hindered their applications using these conjugation methods. Bio-orthogonal “click” reactions usually provide high yields of the targeted conjugates, however, the introduction of azide or ethynyl groups at the BODIPY or peptide periphery requires several steps, and it is normally expensive and time consuming.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of BODIPY-Peptide Conjugates for Fluorescence Labeling of EGFR Overexpressing Cells

et al. 2017

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Regioselective functionalization of 2,3,5,6,8-pentachloro-BODIPY 1 produced unsymmetric BODIPY 5, bearing an isothiocyanate group suitable for conjugation, in only four steps. The X-ray structure of 5 reveals a nearly planar BODIPY core with aryl dihedral angles in the range 47.4° – 62.9°. Conjugation of 5 to two EGFR-targeting pegylated peptides, 3PEG-LARLLT (6) and 3PEG-GYHWYGYTPQNVI (7), under mild conditions (30 min at room temperature), afforded BODIPY conjugates 8 and 9 in 50–80% isolated yields. These conjugates show red-shifted absorption and emission spectra compared with 5, in the near-IR region, and were evaluated as potential fluorescence imaging agents for EGRF over-expressing cells. SPR and docking investigations suggested that conjugate 8 bearing the LARLLT sequence binds to EGFR more effectively than 9 bearing the GYHWYGYTPQNVI peptide, in part due to the lower solubility of 9, and its tendency for aggregation at concentrations above 10 μM. Studies in human carcinoma HEp2 cells over-expressing EGFR, demonstrated low dark and photo-cytotoxicities for BODIPY 5 and the two peptide conjugates, and remarkably high cellular uptake for both conjugates 8 and 9, up to 90-fold compared with BODIPY 5 after 1 h. Fluorescence imaging studies in HEp2 cells revealed subcellular localization of the BODIPY-peptide conjugates mainly in the Golgi apparatus and the cell lysosomes. The low cytotoxicity of the new conjugates and their remarkably high uptake into EGFR over-expressing cells renders them promising imaging agents for cancers over-expressing EGFR.

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Section: Introductionmentioning

confidence: 99%

Synthesis of BODIPY-Peptide Conjugates for Fluorescence Labeling of EGFR Overexpressing Cells

et al. 2017

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“…Consequently, while this technique has enormous potential, it is still in its infancy when it comes to high throughput or exploratory analyses. In addition, the advantage of LSFM in whole-organ imaging is off-set by lower spatial resolution compared to confocal microscopy [12, 38, 39], which enables more refined subcellular analyses [40]. In addition, LSFM of the opaque lung tissue also requires tissue fixation, and thus only allows snapshots of the infection process.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, LSFM of the opaque lung tissue also requires tissue fixation, and thus only allows snapshots of the infection process. In contrast, multiphoton microscopy allows for live imaging of explanted organs [39, 41–43] or even in vivo microscopy [44, 45] using a thoracic window [46] even if a smaller field of view. Fortunately, as recently demonstrated [11], LSFM and two-photon microscopy can complement each other with high added value so that both tissue architecture and cell dynamics can be interrogated to investigate the dynamic temporal host-pathogen interactions as well as its large-scale distributions.…”

Section: Discussionmentioning

confidence: 99%

3D light sheet fluorescence microscopy of lungs to dissect local host immune -Aspergillus fumigatusinteractions

Amich

Mokhtari

Strobel

et al. 2019

Preprint

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Rudolf-Virchow-Zentrum für Experimentelle Biomedizin. 35 Universität Würzburg. 36 Josef-Schneider-Straße 2, Haus D15. D -97080 Würzburg 37 38 39 40 41 42 43 44 45 VISUAL ABSTRACT 46 Quantitative light sheet fluorescence microscopy to dissect local host-pathogen interactions 47 ABSTRACT 51Aspergillus fumigatus is an opportunistic fungal pathogen that can cause life-threatening 52 invasive lung infections in immunodeficient patients. The cellular and molecular processes of 53 infection during onset, establishment and progression are highly complex and depend on 54 both fungal attributes and the immune status of the host. Therefore, preclinical animal 55 models are paramount to investigate and gain better insight into the infection process. Yet, 56 despite their extensive use, commonly employed murine models of invasive pulmonary 57 aspergillosis are not well understood due to analytical limitations. Here we present 58 quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the 59 local immune response in whole lungs at cellular resolution within its anatomical context. We 60 analyzed three very common murine models of pulmonary aspergillosis based on 61 immunosuppression with corticosteroids, chemotherapy-induced leukopenia or 62 myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and 63 degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial 64 distribution, interaction and activation of two key immune cell populations in antifungal 65 defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the 66 patterns of fungal growth correlated with the detected effects of the immunosuppressive 67 regimens on the local immune cell populations. Moreover, LSFM demonstrates that the 68 commonly used intranasal route of spore administration did not result in the desired intra-69 alveolar deposition, as more than 60% of fungal growth occurred outside of the alveolar 70 space. Hence, LSFM allows for more rigorous characterization of murine models of invasive 71 pulmonary aspergillosis and pinpointing their strengths and limitations. 72 IMPORTANCE 73The use of animal models of infection is essential to advance our understanding of complex 74 host-pathogen interactions that take place during Aspergillus fumigatus lung infections. As in 75 the case of humans, mice need to be immunosuppressed to become susceptible to invasive 76 pulmonary aspergillosis, the most serious infection caused by A. fumigatus. There are 77 several immunosuppressive regimens that are routinely used to investigate fungal growth 78 and/or immune responses in murine models of invasive pulmonary aspergillosis (IPA). 79However, the precise consequences that each immunosuppressive model has on the local 80 immune populations and for fungal growth are not completely understood. Here we 81 employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular 82 resolution, to pin down the scenario commonly used IPA models. Our results will be ...

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“…In this context, we have recently described the straightforward synthesis of a fluorogenic tryptophan (Trp) based amino acid as a key building block for the preparation of peptide‐based fluorophores 22. The amino acid was prepared in a single step and in good yields by coupling meta ‐iodophenyl‐substituted BODIPY and Fmoc‐Trp‐OH in the presence of Pd(OAc) 2 under microwave irradiation (Figure 4, top).…”

Section: Metal‐catalyzed C−h Activation Reactionsmentioning

confidence: 99%

Modern Synthetic Avenues for the Preparation of Functional Fluorophores

et al. 2017

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Biomedical research relies on the fast and accurate profiling of specific biomolecules and cells in a non‐invasive manner. Functional fluorophores are powerful tools for such studies. As these sophisticated structures are often difficult to access through conventional synthetic strategies, new chemical processes have been developed in the past few years. In this Minireview, we describe the most recent advances in the design, preparation, and fine‐tuning of fluorophores by means of multicomponent reactions, C−H activation processes, cycloadditions, and biomolecule‐based chemical transformations.

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Spacer-free BODIPY fluorogens in antimicrobial peptides for direct imaging of fungal infection in human tissue

Cited by 116 publications

References 64 publications

Synthesis of BODIPY-Peptide Conjugates for Fluorescence Labeling of EGFR Overexpressing Cells

Synthesis of BODIPY-Peptide Conjugates for Fluorescence Labeling of EGFR Overexpressing Cells

3D light sheet fluorescence microscopy of lungs to dissect local host immune -Aspergillus fumigatusinteractions

Modern Synthetic Avenues for the Preparation of Functional Fluorophores

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