Design, synthesis and characterization of peptidomimetic conjugate of BODIPY targeting HER2 protein extracellular domain

Banappagari, Sashikanth; McCall, Alecia; Fontenot, Krystal R.; Vicente, M. Graça H.; Gujar, Amit D.; Satyanarayanajois, Seetharama D.

doi:10.1016/j.ejmech.2013.04.038

Cited by 22 publications

(26 citation statements)

References 43 publications

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“…The sdAb 5F7 competes with trastuzumab, which binds to domain IV of HER2, which is closest to the cell membrane [40]. On the other hand, 2Rs15d does not compete with trastuzumab or another clinically utilized anti-HER2 therapeutic antibody pertuzumab, which binds to the central region of domain II of HER2 [41].…”

Section: Discussionmentioning

confidence: 99%

Fluorine-18 Labeling of the HER2-Targeting Single-Domain Antibody 2Rs15d Using a Residualizing Label and Preclinical Evaluation

et al. 2017

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Purpose Our previous studies with F-18-labeled anti-HER2 single-domain antibodies (sdAbs) utilized 5F7, which binds to the same epitope on HER2 as trastuzumab, complicating its use for positron emission tomography (PET) imaging of patients undergoing trastuzumab therapy. On the other hand, sdAb 2Rs15d binds to a different epitope on HER2 and thus might be a preferable vector for imaging in these patients. The aim of this study was to evaluate the tumor targeting of F-18 -labeled 2Rs15d in HER2-expressing breast carcinoma cells and xenografts. Procedures sdAb 2Rs15d was labeled with the residualizing labels N-succinimidyl 3-((4-(4-[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([18F]RL-I) and N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB), and the purity and HER2-specific binding affinity and immunoreactivity were assessed after labeling. The biodistribution of I-125- and F-18-labeled 2Rs15d was determined in SCID mice bearing subcutaneous BT474M1 xenografts. MicroPET/x-ray computed tomograph (CT) imaging of [18F]RL-I-2Rs15d was performed in this model and compared to that of nonspecific sdAb [18F]RL-I-R3B23. MicroPET/CT imaging was also done in an intracranial HER2-positive breast cancer brain metastasis model after administration of 2Rs15d-, 5F7-, and R3B23-[18F]RL-I conjugates. Results [18F]RL-I was conjugated to 2Rs15d in 40.8 ± 9.1 % yield and with a radiochemical purity of 97–100 %. Its immunoreactive fraction (IRF) and affinity for HER2-specific binding were 79.2 ± 5.4 % and 7.1 ± 0.4 nM, respectively. [125I]SGMIB was conjugated to 2Rs15d in 58.4 ± 8.2 % yield and with a radiochemical purity of 95–99 %; its IRF and affinity for HER2-specific binding were 79.0 ± 12.9 % and 4.5 ± 0.8 nM, respectively. Internalized radioactivity in BT474M1 cells in vitro for [18F]RL-I-2Rs15d was 43.7 ± 3.6, 36.5 ± 2.6, and 21.7 ± 1.2 % of initially bound radioactivity at 1, 2, and 4 h, respectively, and was similar to that seen for [125I]SGMIB-2Rs15d. Uptake of [18F]RL-I-2Rs15d in subcutaneous xenografts was 16–20 %ID/g over 1–3 h. Subcutaneous tumor could be clearly delineated by microPET/CT imaging with [18F]RL-I-2Rs15d but not with [18F]RL-I-R3B23. Intracranial breast cancer brain metastases could be visualized after intravenous administration of both [18F]RL-I-2Rs15d and [18F]RL-I-5F7. Conclusions Although radiolabeled 2Rs15d conjugates exhibited lower tumor cell retention both in vitro and in vivo than that observed previously for 5F7, given that it binds to a different epitope on HER2 from those targeted by the clinically utilized HER2-targeted therapeutic antibodies trastuzumab and pertuzumab, F-18-labeled 2Rs15d has potential for assessing HER2 status by PET imaging after trastuzumab and/or pertuzumab therapy.

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Section: Discussionmentioning

confidence: 99%

Fluorine-18 Labeling of the HER2-Targeting Single-Domain Antibody 2Rs15d Using a Residualizing Label and Preclinical Evaluation

et al. 2017

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“…By comparison, antibodies can stimulate the HER2 pathway and potentially accelerate cell proliferation and tumorigenesis. 45-50 …”

Section: Discussionmentioning

confidence: 99%

Design and Synthesis of Near-Infrared Peptide for in Vivo Molecular Imaging of HER2

et al. 2015

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We report the development, characterization, and validation of a peptide specific for the extracellular domain of HER2. This probe chemistry was developed for molecular imaging by using a structural model to select an optimal combination of amino acids that maximize the likelihood for unique hydrophobic and hydrophilic interactions with HER2 domain 3. The sequence KSPNPRF was identified and conjugated with either FITC or Cy5.5 via a GGGSK linker using Fmoc-mediated solid-phase synthesis to demonstrate fiexibility for this chemical structure to be labeled with different fluorophores. A scrambled sequence was developed for control by altering the conformationally rigid spacer and moving both hydrophobic and hydrophilic amino acids on the C-terminus. We validated peptide specificity for HER2 in knockdown and competition experiments using human colorectal cancer cells in vitro, and measured a binding affinity of kd = 21 nM and time constant of k = 0.14 min−1 (7.14 min). We used this peptide with either topical or intravenous administration in a preclinical model of colorectal cancer to demonstrate specific uptake in spontaneous adenomas and to show feasibility for real time in vivo imaging with near-infrared fluorescence. We used this peptide in immunofluorescence studies of human proximal colon specimens to evaluate specificity for sessile serrated and sporadic adenomas. Improved visualization can be used endoscopically to guide tissue biopsy and detect premalignant lesions that would otherwise be missed. Our peptide design for specificity to HER2 is promising for clinical translation in molecular imaging methods for early cancer detection.

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“…mice with A431 tumors). 20,22–27,29–30 Additionally, computational studies 20,25–26 have modeled the binding of these two peptides to the extracellular domain at two different binding sites: EGFR-L2 binds to the EGF binding pocket, while EGFR-L1 binds near domain I, away from the EGF binding pocket.…”

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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Design, synthesis and characterization of peptidomimetic conjugate of BODIPY targeting HER2 protein extracellular domain

Cited by 22 publications

References 43 publications

Fluorine-18 Labeling of the HER2-Targeting Single-Domain Antibody 2Rs15d Using a Residualizing Label and Preclinical Evaluation

Fluorine-18 Labeling of the HER2-Targeting Single-Domain Antibody 2Rs15d Using a Residualizing Label and Preclinical Evaluation

Design and Synthesis of Near-Infrared Peptide for in Vivo Molecular Imaging of HER2

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

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