Journal: Acta Pharmaceutica Sinica. B

Article Title: Enhanced radiotheranostic targeting of integrin α 5 β 1 with PEGylation-enabled peptide multidisplay platform (PEGibody): A strategy for prolonged tumor retention with fast blood clearance

doi: 10.1016/j.apsb.2024.07.006

Figure Lengend Snippet: In vitro binding affinity and stability studies of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303. (A) SPR sensorgrams demonstrating the binding affinity of QM-2301, QM-2302, and QM-2303 for human integrin α 5 β 1 in a concentration-dependent manner. (B) The equilibrium dissociation constant ( Κ D ) of each peptide was calculated based on SPR measurements. The K D values of each precursor are shown. (C, D) Schematic diagram of the binding patterns of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302 (C), and [ 64 Cu]QM-2303 (D) to integrin α 5 β 1. Monomeric [ 64 Cu]QM-2301, and [ 64 Cu]QM-2302 bind to receptors in a single-network pattern. For [ 64 Cu]QM-2303, the PEGibody-based radiotracer, one PEGibody can bind to more than two integrin α 5 β 1 receptors, exhibiting better binding affinity. (E, F) The expression of integrin α 5 β 1 in B16F10 cells was analyzed by flow cytometry (E) and Western blotting (F). For flow cytometry assays, an anti-integrin α 5 + β 1 antibody was used. For Western blot assays, integrin α 5 (∼150 kDa) and integrin β 1 (∼138 kDa) were examined using two antibodies. (G) The stability of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303 after coincubation with mouse serum within 1 h, as indicated by radio-HPLC. (H) I n vitro cell uptake of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303 (750 KBq/mL) when incubated with B16F10 cells for different time period. (I) IC 50 of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303 when inhibited with antibodies at different concentrations. ∗ P < 0.05, ∗∗ P < 0.01. All the quantitative experiments were performed independently at least three times (data are the mean ± SD, n = 3).

Article Snippet: Recombinant human integrin α 5 β 1 (alpha 5 beta 1, HY-P77718, MCE, NJ, USA) was immobilized on a CM5 sensor chip using a standard amine coupling kit at a temperature of 25 °C.

Techniques: In Vitro, Binding Assay, Concentration Assay, Expressing, Flow Cytometry, Western Blot, Incubation

Journal: ACS Omega

Article Title: Design and Evaluation of Short Self-Assembling Depsipeptides as Bioactive and Biodegradable Hydrogels

doi: 10.1021/acsomega.7b01641

Figure Lengend Snippet: Half inhibition (i.e., IC 50 ) of the fluorescence of FITC-GRGDSP occurs at a ∼40-fold lower concentration for RGD than R-Glc-D, indicating greater affinity of RGD for integrin than R-Glc-D (A). Comparison of FP inhibition by R-Glc-D and RGE peptides (negative control). The average IC 50 value for R-Glc-D is 1115 ± 211 μM (mean ± standard deviation). RGE was tested once as a negative control, and the calculated IC 50 value was 2840 μM (B).

Article Snippet: Human recombinant integrin α 5 β 1 (R&D Systems) was diluted to 900 nM in the same Tris buffer.

Techniques: Inhibition, Fluorescence, Concentration Assay, Comparison, Negative Control, Standard Deviation

Journal: ChemistryOpen

Article Title: Insights into the Binding of Cyclic RGD Peptidomimetics to α 5 β 1 Integrin by using Live‐Cell NMR And Computational Studies

doi: 10.1002/open.201600112

Figure Lengend Snippet: Docking best poses of a) compound 7 (green) and b) compound 6 (green) in the crystal structure of the extracellular domain of α 5 β 1 integrin (α 5 subunit pink, β 1 subunit cyan, model from 3VI4.pdb). Only selected integrin residues involved in interactions with the ligand are shown. Non polar hydrogens are hidden for clarity, whereas intermolecular hydrogen bonds are shown as dashed lines.

Article Snippet: Purified recombinant human integrin α 5 β 1 (R&D Systems, Inc., Minneapolis, MN, USA) was diluted to 0.5 μg mL −1 in coating buffer containing 20 m m Tris‐HCl (pH 7.4), 150 m m NaCl, 1 m m MnCl 2 , 2 m m CaCl 2 , and 1 m m MgCl 2 .

Techniques:

Journal: ChemistryOpen

Article Title: Insights into the Binding of Cyclic RGD Peptidomimetics to α 5 β 1 Integrin by using Live‐Cell NMR And Computational Studies

doi: 10.1002/open.201600112

Figure Lengend Snippet: Docking binding modes: a) A and b) B of compound 3 (green) in the crystal structure of the extracellular domain of α 5 β 1 integrin (α 5 subunit pink, β 1 subunit cyan, model from 3VI4.pdb). Only selected integrin residues involved in interactions with the ligand are shown. Non polar hydrogens are hidden for clarity, whereas intermolecular hydrogen bonds are shown as dashed lines.

Article Snippet: Purified recombinant human integrin α 5 β 1 (R&D Systems, Inc., Minneapolis, MN, USA) was diluted to 0.5 μg mL −1 in coating buffer containing 20 m m Tris‐HCl (pH 7.4), 150 m m NaCl, 1 m m MnCl 2 , 2 m m CaCl 2 , and 1 m m MgCl 2 .

Techniques: Binding Assay

Journal: ChemistryOpen

Article Title: Insights into the Binding of Cyclic RGD Peptidomimetics to α 5 β 1 Integrin by using Live‐Cell NMR And Computational Studies

doi: 10.1002/open.201600112

Figure Lengend Snippet: Inhibition of biotinylated fibronectin binding to α 5 β 1 integrin compared with inhibition of biotinylated vitronectin binding to α v β 3 .

Article Snippet: Purified recombinant human integrin α 5 β 1 (R&D Systems, Inc., Minneapolis, MN, USA) was diluted to 0.5 μg mL −1 in coating buffer containing 20 m m Tris‐HCl (pH 7.4), 150 m m NaCl, 1 m m MnCl 2 , 2 m m CaCl 2 , and 1 m m MgCl 2 .

Techniques: Inhibition, Binding Assay

Journal:

Article Title: The EphA8 Receptor Regulates Integrin Activity through p110? Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

doi: 10.1128/MCB.21.14.4579-4597.2001

Figure Lengend Snippet: (A) Effects of anti-α5β1 and anti-β3 integrin blocking antibodies (Ab) on NIH 3T3 cell adhesion to fibronectin. NIH 3T3 fibroblasts stably expressing wild-type or kinase-inactive EphA8 were detached and incubated for 5 min with either anti-α5β1 or anti-β3 integrin blocking antibody (5 μg/ml). The cells were then replated on fibronectin-coated coverslips and allowed to adhere for 15 min. Data from three independent experiments are presented as means ± SE. (B) Induction of wild-type EphA8 protein expression in NIH 3T3 fibroblasts. Cells were incubated with 2 μg of doxycycline (Dox) per ml for the indicated periods of time and then lysed, and proteins from the lysates were immunoprecipitated with anti-EphA8 antibody. Immunoprecipitates were separated by 7.5% SDS-PAGE and immunoblotted with anti-EphA8 antibody (top); the same blot was stripped of antibodies and reprobed with antiphosphotyrosine antibody (bottom). (C) Effects of anti-α5β1 and anti-β3 integrin blocking antibodies on NIH 3T3 cell attachment stimulated by the induced expression of EphA8. Cells were treated with doxycycline for the indicated periods of time, and cell attachment assays were performed after incubation with either anti-α5β1 or anti-β3 blocking antibodies as described above. (D) Analysis of α5β1 and β3 integrins expressed in NIH 3T3 cells, concomitant with the induced expression of EphA8. EphA8 expression was induced for the indicated times, then the cells were harvested and cell surface proteins were biotinylated; integrins were immunoprecipitated with anti-α5 polyclonal antibody, and biotinylated integrins were detected using streptavidin-HRP (top). Cells were lysed in PLC lysis buffer, and protein concentrations were equalized (bottom). Cell lysates were fractionated by SDS-PAGE and then analyzed by immunobloting using anti-β3 antibody as a probe.

Article Snippet: Monoclonal anti-human integrin α 5 β 1 (JBS5) and murine α 5 β 1 (BMA5) and polyclonal anti-murine integrin α 5 antibodies were from Chemicon.

Techniques: Blocking Assay, Stable Transfection, Expressing, Incubation, Immunoprecipitation, SDS Page, Cell Attachment Assay, Lysis, Western Blot

Journal:

Article Title: The EphA8 Receptor Regulates Integrin Activity through p110? Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

doi: 10.1128/MCB.21.14.4579-4597.2001

Figure Lengend Snippet: (A) Induction of wild-type EphA8 protein expression in HEK293 epithelial cells. Cells were incubated with 2 μg of doxycycline (Dox) per ml for the indicated times. Proteins from cell lysates were immmunoprecipitated with anti-EphA8 antibody, then separated by 7.5% SDS-PAGE, and immunoblotted with the same antibody (top); the same blot was stripped and then reprobed with antiphosphotyrosine antibody (middle); Bottom, analysis of α5β1 integrin expressed in HEK293 cells, concomitant with the induced expression of EphA8. EphA8 expression was induced for the indicated times, then the cells were harvested, and cell surface proteins were biotinylated. Labeled integrins were immunoprecipitated with anti-α5β1 monoclonal antibody and then detected with streptavidin-HRP. (B) Effect of anti-α5β1 integrin blocking antibodies (Ab) on the cell attachment stimulated by the induced expression of EphA8 in HEK293 cells. The cells were treated with doxycycline for the indicated times, and then cell attachment assays were performed after incubation with anti-α5β1 blocking antibodies as described in the legend to Fig. ​Fig.2A.2A. Note that HEK293 cells were allowed to adhere for 30 min before being washed to remove nonadherent cells, and then incubation was continued for 90 min. The percentage of cells attached after 30 min is shown. Data from three independent experiments are presented as means ± SE. (C) Induction of kinase-inactive EphA8 protein expression in HEK293 epithelial cells. As a positive control, HEK293 cells inducibly expressing the wild-type EphA8 protein were treated with doxycycline for 12 h. Proteins from cell lysates were immmunoprecipitated with anti-EphA8 antibody, then separated by 7.5% SDS-PAGE, and immunoblotted with anti-EphA8 antibody (top); the same blot was stripped and then reprobed with antiphosphotyrosine antibody (bottom). (D) Effects of anti-α5β1 integrin blocking antibodies on cell attachment stimulated by the induced expression of kinase-inactive EphA8 receptor in HEK293 cells. Cell attachment assays were performed as described for panel B.

Article Snippet: Monoclonal anti-human integrin α 5 β 1 (JBS5) and murine α 5 β 1 (BMA5) and polyclonal anti-murine integrin α 5 antibodies were from Chemicon.

Techniques: Expressing, Incubation, SDS Page, Labeling, Immunoprecipitation, Blocking Assay, Cell Attachment Assay, Positive Control

Journal:

Article Title: The EphA8 Receptor Regulates Integrin Activity through p110? Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

doi: 10.1128/MCB.21.14.4579-4597.2001

Figure Lengend Snippet: (A) Transient expression and tyrosine phosphorylation of EphA8 point mutants and kinase-inactive proteins. HEK293 cells were transiently transfected with pcDNA3-derived expression plasmids containing the recombinant cDNAs encoding the indicated EphA8 mutant proteins and were lysed for immunoprecipitation with anti-EphA8 antibody at 24 h posttransfection. Western blot analysis was performed with anti-EphA8 antibody to verify the level of EphA8 expression (top); the same blot was stripped and then reprobed with antiphosphotyrosine antibody to evaluate the level of autophosphorylation of the transfected EphA8 mutant proteins (bottom). (B) Mutations of two major tyrosine phosphorylation sites and the ATP binding site lysine of the EphA8 receptor do not affect EphA8-stimulated cell attachment to fibronectin. Cell attachment assays were performed as described in previous figure legends. (C) Top, schematic representation of the EphA8 cytoplasmic domain deletion mutants. Middle, HEK293 cells were transiently transfected with the indicated constructs, and cells were lysed for immunoprecipitation with anti-HA polyclonal antibody at 24 h posttransfection. Western blot analysis was performed with same antibody to verify the expression level of the deletion mutant. Note that the anti-EphA8 antibody does not recognize the EphA8 mutant lacking the JM encoded by exon X. Bottom, the same blot was stripped and then reprobed with antiphosphotyrosine antibody. (D) Cell attachment assays performed as described in previous figure legends.

Article Snippet: Monoclonal anti-human integrin α 5 β 1 (JBS5) and murine α 5 β 1 (BMA5) and polyclonal anti-murine integrin α 5 antibodies were from Chemicon.

Techniques: Expressing, Transfection, Derivative Assay, Recombinant, Mutagenesis, Immunoprecipitation, Western Blot, Binding Assay, Cell Attachment Assay, Construct

Journal:

Article Title: The EphA8 Receptor Regulates Integrin Activity through p110? Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

doi: 10.1128/MCB.21.14.4579-4597.2001

Figure Lengend Snippet: (A) Inhibition of EphA8-stimulated cell attachment by wortmannin. Expression of wild-type or kinase-inactive EphA8 proteins in HEK293 cells was induced with doxycycline (Dox) for 24 h, and cells were incubated with wortmannin at concentrations ranging from 10 to 100 nM for 30 min prior to cell attachment assays. Cells that had not been treated with doxycycline were included as controls. Wortmannin was dissolved in DMSO. Note that cells were treated with DMSO for 30 min prior to the cell attachment assay in the absence of wortmannin (0 nM on the x axis). Treatment of cells with DMSO (5 to 10 μl) had no effect on the basal level of adhesion or EphA8-stimulated cell adhesion. (B) The EphA8 proteins were induced with doxycycline for the indicated times and then immunoprecipitated with either anti-HA antibody (top, left and middle) or antiphosphotyrosine antibody (top, right). Immunoprecipitates were incubated with 4 μg of PI and 10 μCi of [γ-32P]ATP for 10 min, and the reaction was analyzed by TLC followed by autoradiography. Western blot analysis of anti-HA immunoprecipitates was performed with the same antibody to verify the expression levels of wild-type and kinase-inactive EphA8 proteins at the indicated induction times (bottom). (C and D) The EphA8 JM is necessary for association with PI 3-kinase activity. HEK293 cells were transiently transfected with the indicated constructs, and cells were lysed for immunoprecipitation of the EphA8 deletion mutants with anti-HA polyclonal antibody at 24 h posttransfection. Western blot analysis was performed with same antibody to verify the expression level of the deletion mutant (C, bottom) PI 3-kinase activity was determined by TLC and autoradiography (C, top) and also by measuring formation of radiolabeled PIP from PI, using a phosphorimaging system (D). Shown are mean values (±SE) of three independent experiments. (E) p85-dependent heterodimeric PI 3-kinase is not stimulated to response to EphA8 expression. The kinase-inactive EphA8 protein was induced with doxycycline for the indicated times, and then p85-p110 heterodimeric PI 3-kinase was immunoprecipitated with anti-p85 polyclonal antibody. As a control, the same cells were starved of serum for 24 h and then stimulated with (+) or without (−) serum for 15 min in the absence of doxycycline. Immunoprecipitates were analyzed by in vitro PI 3-kinase assay, and lipids were extracted and analyzed by TLC and autoradiography as described above.

Article Snippet: Monoclonal anti-human integrin α 5 β 1 (JBS5) and murine α 5 β 1 (BMA5) and polyclonal anti-murine integrin α 5 antibodies were from Chemicon.

Techniques: Inhibition, Cell Attachment Assay, Expressing, Incubation, Immunoprecipitation, Autoradiography, Western Blot, Activity Assay, Transfection, Construct, Mutagenesis, In Vitro, Kinase Assay

Journal:

Article Title: The EphA8 Receptor Regulates Integrin Activity through p110? Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

doi: 10.1128/MCB.21.14.4579-4597.2001

Figure Lengend Snippet: Evidence that p110γ PI 3-kinase is tightly associated with the EphA8 receptor. (A) Left, HEK293 cells were transfected with the Δp85 cDNA, and individual G418-resistant clones were isolated. A representative cell line was treated with (+) or without (−) serum for 15 min, cells were lysed in PLC lysis buffer, and p110-p85 heterodimers from the cell lysates were immmuprecipitated with anti-p85 polyclonal antibody (lanes 1 and 2). The stable Δp85 transfectants were transiently transfected with the indicated constructs and lysed for immunoprecipitation of the EphA8 proteins with anti-HA polyclonal antibody at 24 h posttransfection (lanes 3 to 5). PI-3 kinase activity was measured as described in previous figure legends. Right, cell attachment assays performed as in previous figure legends. (B) EphA8 protein expression in HEK293 cells was induced with doxycycline treatment for the indicated times, and then PI 3-kinases were immunoprecipitated (IP) with the indicated anti-p110 isotype antibodies. PI 3-kinase activity was measured as described in previous figure legends. (C) Expression of wild-type or kinase-inactive EphA8 proteins in HEK293 cells was induced with doxycycline for the indicated times. Top, proteins from cell lysates were immunoprecipitated with anti-p110γ antibody and then analyzed by immunoblot using anti-EphA8 antibody as a probe (top); the same blot was stripped and reprobed with anti-p110γ antibody (bottom).

Article Snippet: Monoclonal anti-human integrin α 5 β 1 (JBS5) and murine α 5 β 1 (BMA5) and polyclonal anti-murine integrin α 5 antibodies were from Chemicon.

Techniques: Transfection, Clone Assay, Isolation, Lysis, Construct, Immunoprecipitation, Activity Assay, Cell Attachment Assay, Expressing, Western Blot

Journal:

Article Title: The EphA8 Receptor Regulates Integrin Activity through p110? Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

doi: 10.1128/MCB.21.14.4579-4597.2001

Figure Lengend Snippet: The JM deletion EphA8 mutant suppresses EphA8-promoted p110γ PI 3-kinase activity and integrin activation by inhibiting the association of EphA8 with p110γ. HEK293 cells expressing EphA8 in response to doxycycline (Dox) were transiently transfected with the indicated constructs; 12 h after transfection, EphA8 protein expression was induced for 12 h. (A) Cell attachment assays performed as described in previous figure legends. (B) Proteins from cell lysates were immunoprecipitated with anti-p110γ antibody, and then PI-3 kinase activity was measured as described in previous figure legends. (C) Proteins from cell lysates were immunoprecipitated with anti-p110γ antibody and then analyzed by immunoblotting with anti-EphA8 antibody as a probe (top); the same blot was stripped and reprobed with anti-HA and anti-p110γ antibodies (middle and bottom, respectively). (D) The EphA8 JM is sufficient for association with p110γ. A FLAG-tagged murine p110γ construct was transiently transfected into parental cells (lane 5) or HEK293 cells expressing EphA8 in response to doxycycline treatment (lanes 1 to 4). As a control, the FLAG-tagged p110γ protein transiently expressed in parental HEK293 cells was directly immunoprecipitated with anti-FLAG antibody (lane 5). The induction of EphA8 began 12 h after transfection and continued for 24 h (lanes 2 and 4). Proteins from each cell lysate were mixed with approximately equal amounts of purified GST or with GST-JM fusion protein bound to glutathione-Sepharose beads (lanes 1 to 4). The washed beads were separated by 7.5% SDS-PAGE and Western blotted using anti-FLAG antibody as a probe. (E) The EphA8 JM is sufficient for direct association with p110γ. HEK293 cells expressing both exogenous p110γ and wild-type EphA8 were prepared as described for panel D. FLAG-tagged p110γ was directly immunoprecipitated with anti-FLAG antibody, and then the washed beads were directly separated by 7.5% SDS-PAGE and transferred to membranes. The blots were probed with 32P-labeled GST-JM protein to detect p110γ. The labeling of GST-JM protein was performed in an in vitro kinase assay with [γ-32P]ATP and anti-HA immunoprecipitates containing wild-type EphA8 as previously described. Note that the GST-JM fusion protein contained Tyr-615, which is a major phosphorylation site.

Article Snippet: Monoclonal anti-human integrin α 5 β 1 (JBS5) and murine α 5 β 1 (BMA5) and polyclonal anti-murine integrin α 5 antibodies were from Chemicon.

Techniques: Mutagenesis, Activity Assay, Activation Assay, Expressing, Transfection, Construct, Cell Attachment Assay, Immunoprecipitation, Western Blot, Purification, SDS Page, Labeling, In Vitro, Kinase Assay