Journal: Journal of Medicinal Chemistry

Article Title: Innately Fluorescent Tetravalent Cytotoxic Conjugate TetraF HER2 -vcMMAE Engages Aggregation-Dependent Endocytosis of HER2 for Enhanced Intracellular Drug Delivery

doi: 10.1021/acs.jmedchem.5c00782

Figure Lengend Snippet: Engineering of fluorescent, multivalent HER2-specific ligands. (A) Schematic representation of the engineered fluorescent multivalent GFPp_Affibody HER2:342 oligomers. The mixture of GFPp_Affibody HER2:342 oligomers was purified by affinity chromatography and analyzed using SDS-PAGE (B) and Western blotting with anti-His-Tag antibodies. Asterisks mark nonfully denatured higher oligomeric forms of multivalent HER2 ligands in (C). (D) The identity of GFPp_Affibody HER2:342 was confirmed by mass spectrometry. (E) The mixture of GFPp_Affibody HER2:342 oligomers was separated under nondenaturing conditions by Native PAGE. The fluorescence properties of the purified oligomers were assessed by UV imaging of Native PAGE gels. (F) The efficacy of isolating different oligomeric forms was confirmed by Native PAGE and Western blotting with anti-His-Tag antibodies. (G) The oligomeric state of the purified proteins was assessed by size exclusion chromatography (SEC). The slight differences in the masses are because, in this technique, the separation of the oligomers is also influenced by their shape.

Article Snippet: The extracellular region of HER2 was immobilized on Protein A sensors and incubated with BiF HER2 , TriF HER2 , TetraF HER2 , and PentaF HER2 or with the monomeric Affibody HER2:342 as a control, and the parameters of the interaction were measured with BLI.

Techniques: Purification, Affinity Chromatography, SDS Page, Western Blot, Mass Spectrometry, Clear Native PAGE, Fluorescence, Imaging, Size-exclusion Chromatography

Journal: Journal of Medicinal Chemistry

Article Title: Innately Fluorescent Tetravalent Cytotoxic Conjugate TetraF HER2 -vcMMAE Engages Aggregation-Dependent Endocytosis of HER2 for Enhanced Intracellular Drug Delivery

doi: 10.1021/acs.jmedchem.5c00782

Figure Lengend Snippet: Stability analysis of multivalent HER2-specific ligands. The oligomers were incubated in a serum-free medium at 37 °C for 96 h. At distinct time points, samples were taken, and the stability of the GFPp_Affibody HER2:342 oligomers was analyzed by SDS-PAGE (A) and UV imaging of Native PAGE gels (B). (C) The stability of the GFPp oligomerization scaffold was confirmed by monitoring GFP fluorescence emission at different points of incubation of the oligomers in 10-fold diluted human serum at 37 °C. Fluorescence spectra were acquired with excitation at 488 nm and emission in the 500–650 nm range. Representative data from three independent experiments are shown.

Article Snippet: The extracellular region of HER2 was immobilized on Protein A sensors and incubated with BiF HER2 , TriF HER2 , TetraF HER2 , and PentaF HER2 or with the monomeric Affibody HER2:342 as a control, and the parameters of the interaction were measured with BLI.

Techniques: Incubation, SDS Page, Imaging, Clear Native PAGE, Fluorescence

Journal: Journal of Medicinal Chemistry

Article Title: Innately Fluorescent Tetravalent Cytotoxic Conjugate TetraF HER2 -vcMMAE Engages Aggregation-Dependent Endocytosis of HER2 for Enhanced Intracellular Drug Delivery

doi: 10.1021/acs.jmedchem.5c00782

Figure Lengend Snippet: Analysis of the affinity of multivalent ligands for HER2. (A) The expression level of HER2 in SKBR-3 and MCF-7 cell lines was analyzed by Western blotting. Tubulin levels were used as loading control. (B) The specificity of the interaction between GFPp_Affibody HER2:342 oligomers and HER2 receptor was confirmed by confocal microscopy. SKBR-3 and MCF-7 cells were incubated with 300 nM BiF HER2 , TriF HER2 , TetraF HER2 , and PentaF HER2 for 30 min on ice. Nuclei were stained with NucBlue Live dye, and cells were fixed in a 4% paraformaldehyde solution. (C) GFPp_Affibody HER2:342 oligomers were incubated with the recombinant extracellular domain of HER2 (HER2.ecd-Fc) for 15 min at room temperature, and the formation of the ligand–receptor complex was confirmed by Native PAGE and Western blotting using anti-HER2 antibodies. Oligomers without added receptors were used as a control. (D) BLI analyses of the interaction between GFPp_Affibody HER2:342 oligomers and the HER2 receptor. HER2-Fc was immobilized on Protein A sensors, and the association and dissociation phases were monitored at different protein concentrations. A reference sensor without HER2-Fc was used as a control. (E) Microscopic analysis of the enhanced binding of multivalent variants to HER2. SKBR-3 cells were preincubated with DyLight 550-labeled monomeric Affibody HER2:342 for 10 min on ice, then equimolar concentrations of BiF HER2 , TriF HER2 , TetraF HER2 , or PentaF HER2 were added, and incubation was continued for 30 min. Cells incubated with monomeric proteins were used as a control. Nuclei were stained with NucBlue Live dye, and the cells were fixed in a 4% paraformaldehyde solution. Representative images from three independent experiments are shown. The scale bar is 20 μm.

Article Snippet: The extracellular region of HER2 was immobilized on Protein A sensors and incubated with BiF HER2 , TriF HER2 , TetraF HER2 , and PentaF HER2 or with the monomeric Affibody HER2:342 as a control, and the parameters of the interaction were measured with BLI.

Techniques: Expressing, Western Blot, Control, Confocal Microscopy, Incubation, Staining, Recombinant, Clear Native PAGE, Binding Assay, Labeling

Journal: Journal of Medicinal Chemistry

Article Title: Innately Fluorescent Tetravalent Cytotoxic Conjugate TetraF HER2 -vcMMAE Engages Aggregation-Dependent Endocytosis of HER2 for Enhanced Intracellular Drug Delivery

doi: 10.1021/acs.jmedchem.5c00782

Figure Lengend Snippet: Efficient clustering-based endocytosis of multivalent HER2 ligands. (A) The internalization of BiF HER2 , TriF HER2 , TetraF HER2 , and PentaF HER2 into SKBR-3 cells was analyzed by using quantitative confocal microscopy. Cells were treated with oligomers for 30 min at 37 °C. Nuclei were stained with NucBlue Live dye, and the cells were fixed, permeabilized with 0.1% Triton in PBS, and stained with HCS CellMask Deep Red Stain. Representative images from three independent experiments are shown. The scale bar represents 20 μm. Each gray spot in the graph represents the relative intracellular punctate signal intensity oligomers in the single cell. At least 200 cells for each condition from three independent experiments were measured. Horizontal lines in the graph represent the average intensity of the intracellular oligomer punctate signal, whereas boxes represent ± SD. Statistical analyses were performed using analysis of variance (ANOVA) with Tukey HSD for unequal N (Spjotvoll/Stoline) posthoc test (* p < 0.05; ** p < 0.005 and *** p < 0.001). (B) Colocalization of TetraF HER2 with early endosome marker EEA1. SKBR-3 cells were incubated with TetraF HER2 for 30 min at 37 °C. Early endosomes were detected with rabbit polyclonal antibody specific for early endosome antigen 1 (EEA1) and antirabbit IgG secondary antibody conjugated to Alexa Fluor 594 (red). Scale bars are 20 μm. (C) Colocalization of TetraF HER2 with HER2. SKBR-3 cells were incubated with TetraF HER2 for 30 min at 37 °C. HER2 was detected with mouse monoclonal antibody specific for HER2 (ErbB2/HER2) and antimouse IgG secondary antibody conjugated to Alexa Fluor 594 (red). Scale bars represent 20 μm. (D) DLS signals of TetraF HER2 , HER2, and mixtures of these proteins. DLS-estimated MW of the proteins are shown. High molecular weight complexes are seen upon incubation of TetraF HER2 and HER2. (E) Western blotting analysis of cell lysates of SKBR-3 cells treated with siRNA against clathrin heavy chain (CLTC), dynamin-2 (DNM2), and scramble siRNA as a control. CBB was used as a loading control. (F) Analysis of the effect of the depletion of CLTC and DNM2 on the endocytosis of TetraF HER2 . SKBR-3 cells after CLTC and DNM2 knock-down were incubated with TetraF HER2 for 30 min at 37 °C, and internalization was analyzed using quantitative confocal microscopy. Representative images from three independent experiments are shown. The scale bar represents 20 μm. Each gray spot in the graph represents the relative intracellular punctate signal intensity of the TetraF HER2 in the single cell. At least 200 cells for each condition from three independent experiments were measured. Horizontal lines in the graph represent the average intensity of the intracellular TetraF HER2 punctate signal, whereas boxes represent ± SD. Statistical analyses were performed using analysis of variance (ANOVA) with Tukey HSD for unequal N (Spjotvoll/Stoline) posthoc test (* p < 0.05; ** p < 0.005 and *** p < 0.001).

Article Snippet: The extracellular region of HER2 was immobilized on Protein A sensors and incubated with BiF HER2 , TriF HER2 , TetraF HER2 , and PentaF HER2 or with the monomeric Affibody HER2:342 as a control, and the parameters of the interaction were measured with BLI.

Techniques: Confocal Microscopy, Staining, Marker, Incubation, High Molecular Weight, Western Blot, Control, Knockdown

Journal: Journal of Medicinal Chemistry

Article Title: Innately Fluorescent Tetravalent Cytotoxic Conjugate TetraF HER2 -vcMMAE Engages Aggregation-Dependent Endocytosis of HER2 for Enhanced Intracellular Drug Delivery

doi: 10.1021/acs.jmedchem.5c00782

Figure Lengend Snippet: Development of an inherently fluorescent tetrameric cytotoxic conjugate against HER2-overexpressing breast cancer cells. (A) Schematic representation of the conjugation reaction and fluorescent tetrameric TetraF HER2 -vcMMAE conjugate. Sortase A recognizes the LPETGG sequence within the tetrameric protein and mediates ligation of the peptide-linked MMAE, resulting in TetraF HER2 -vcMMAE. The correctness of the synthesized GGGS-(O2Oc) 2 -C-NH 2 peptide (B) and its conjugation with the vcMMAE (C) was confirmed with mass spectrometry. (D) The successful attachment of drug to TetraF HER2 was confirmed with SDS-PAGE, Western blotting, and anti-MMAE antibodies (E) and with mass spectrometry (F) asterisks in E mark nonfully denatured distinct oligomeric forms of the conjugate. (G) The cytotoxic effect of TetraF HER2 -vcMMAE was tested on MCF-7, MDA-MB-231, MDA-MB-453, BT-474 and SKBR-3 cells. Cells were treated with increasing concentrations of TetraF HER2 , TetraF HER2 -vcMMAE, and free drug at 37 °C for 96 h. Cell viability was analyzed with the PrestoBlue Cell Viability Reagent. Data shown are the mean values of three independent experiments ± SD. Statistical analyses were performed with Kruskal–Wallis H test (* p < 0.05; ** p < 0.005 and *** p < 0.001). H. The IC50 value of TetraF HER2 -vcMMAE was calculated from Hill’s equation using Origin 7 software.

Article Snippet: The extracellular region of HER2 was immobilized on Protein A sensors and incubated with BiF HER2 , TriF HER2 , TetraF HER2 , and PentaF HER2 or with the monomeric Affibody HER2:342 as a control, and the parameters of the interaction were measured with BLI.

Techniques: Conjugation Assay, Sequencing, Ligation, Synthesized, Mass Spectrometry, SDS Page, Western Blot, Software

Journal: Journal of Medicinal Chemistry

Article Title: Innately Fluorescent Tetravalent Cytotoxic Conjugate TetraF HER2 -vcMMAE Engages Aggregation-Dependent Endocytosis of HER2 for Enhanced Intracellular Drug Delivery

doi: 10.1021/acs.jmedchem.5c00782

Figure Lengend Snippet: Microscopic analysis of the cytotoxic effect of TetraF HER2 -vcMMAE. SKBR-3 and MCF-7 cells were treated with TetraF HER2 , TetraF HER2 -vcMMAE, and free drug at 37 °C for 72 h and imaged in real time using quantitative confocal microscopy. Dead cells were labeled with propidium iodide solution and nuclei of all cells were stained with NucBlue Live dye. Occasional pink color represents overlay of propidium iodide and NucBlue Live staining. Representative images from three independent experiments are shown. The scale bar represents 20 μm. The graph shows the normalized percentages of the intensities of the propidium iodide derived signals. Average values from at three independent experiments ± SD are shown. Statistical analyses were performed using analysis of variance (ANOVA) with the Fishers LSD posthoc test (* p < 0.05; ** p < 0.005, and *** p < 0.001).

Article Snippet: The extracellular region of HER2 was immobilized on Protein A sensors and incubated with BiF HER2 , TriF HER2 , TetraF HER2 , and PentaF HER2 or with the monomeric Affibody HER2:342 as a control, and the parameters of the interaction were measured with BLI.

Techniques: Confocal Microscopy, Labeling, Staining, Derivative Assay