52
2 Synthesis of In-Tether Chiral Center Peptides …
b. MDM 2 /PDI peptide FITC-labeled peptides (10–20 nM) were incubated with
HDM 2 17-125 in binding assay buffer (140 mM NaCl, 50 mM, Tris pH 8.0) at room
temperature for 1 h. Fluorescence polarization experiments were performed in 96well plates (Perkin Elmer Optiplate-96F) on plate reader (Perkin Elmer, Envision,
2104 multilabel reader). Concentrations of the peptides were determined by 494 nm
absorption of FITC. K d values were determined by nonlinear regression analysis of
dose response curves using Origin pro 9.0.
2.4.10 Cell Imaging
HEK 293T cells (or Hela or MCF-7 cells) were cultured with DMEM with 10% FBS
(v/v) in imaging dishes (50,000 cells/well) in 37°C, 5% CO 2 incubator for one day
until they were about 80% adherent. Peptide were first dissolved in DMSO to make
a 1 mM stock and then added to cells to a final concentration of 5 μM. The cells
were incubated with peptides for 1 h at 37°C. After incubation, cells were washed
3 times with PBS and then fixed with 4% formaldehyde (Alfa Aesar, MA) in PBS
for 10 min. They were then washed 3 times with PBS and stained with 1 μg/ml 4’,
6-diamidino-2-phenylindole (DAPI) (Invitrogen, CA) in PBS for 5 min. Images of
peptide localization in cells were taken on PerkinElmer confocal microscopy. Image
processing was done using Volocity software package (Zeiss Imaging).
2.4.11 Flow Cytometry
MCF-7 cells were grown in DMEM medium with 10% FBS (v/v) in imaging dishes
(50,000 cells/well) in 37°C, 5% CO 2 incubator for two days (50,000 cells per well).
Cells were treated with fluoresceinated peptides (5 μM) for up to 2 h at 37°C. After
washing with media, the cells were exposed to trypsin (0.25%; Gibco) digestion
(5 min, 37°C), washed with PBS, and resuspended in PBS. Cellular fluorescence was
analyzed using a BD FACSCalibur flow cytometer (Becton Dickinson) and CellQuest
Pro (or CFlow plus). The identical experiment was performed with 30 min preincubation of cells at 4°C followed by 4 h incubation with fluoresceinated peptides
at 4°C to assess temperature dependence of fluorescent labeling.
2.4.12 Serum Stability
The in vitro serum stability assay followed the procedure of previous literature
[13]. Standard solution of PDI-linear (FITC-ßAla-LTFEHYWAQLTS-NH 2 ), PDI-1b
(FITC-(cyclo-5,9)-[ßAla-LTFCHYWS5(2-Me) (R) QLTS]-NH 2 ), and PDI-2b (FITC(cyclo-5,9)-[ßAla-LTFCHYWS 5 (2-Ph) (R) QLTS]-NH 2 ) was prepared in water. Each
2 Synthesis of In-Tether Chiral Center Peptides …
b. MDM 2 /PDI peptide FITC-labeled peptides (10–20 nM) were incubated with
HDM 2 17-125 in binding assay buffer (140 mM NaCl, 50 mM, Tris pH 8.0) at room
temperature for 1 h. Fluorescence polarization experiments were performed in 96well plates (Perkin Elmer Optiplate-96F) on plate reader (Perkin Elmer, Envision,
2104 multilabel reader). Concentrations of the peptides were determined by 494 nm
absorption of FITC. K d values were determined by nonlinear regression analysis of
dose response curves using Origin pro 9.0.
2.4.10 Cell Imaging
HEK 293T cells (or Hela or MCF-7 cells) were cultured with DMEM with 10% FBS
(v/v) in imaging dishes (50,000 cells/well) in 37°C, 5% CO 2 incubator for one day
until they were about 80% adherent. Peptide were first dissolved in DMSO to make
a 1 mM stock and then added to cells to a final concentration of 5 μM. The cells
were incubated with peptides for 1 h at 37°C. After incubation, cells were washed
3 times with PBS and then fixed with 4% formaldehyde (Alfa Aesar, MA) in PBS
for 10 min. They were then washed 3 times with PBS and stained with 1 μg/ml 4’,
6-diamidino-2-phenylindole (DAPI) (Invitrogen, CA) in PBS for 5 min. Images of
peptide localization in cells were taken on PerkinElmer confocal microscopy. Image
processing was done using Volocity software package (Zeiss Imaging).
2.4.11 Flow Cytometry
MCF-7 cells were grown in DMEM medium with 10% FBS (v/v) in imaging dishes
(50,000 cells/well) in 37°C, 5% CO 2 incubator for two days (50,000 cells per well).
Cells were treated with fluoresceinated peptides (5 μM) for up to 2 h at 37°C. After
washing with media, the cells were exposed to trypsin (0.25%; Gibco) digestion
(5 min, 37°C), washed with PBS, and resuspended in PBS. Cellular fluorescence was
analyzed using a BD FACSCalibur flow cytometer (Becton Dickinson) and CellQuest
Pro (or CFlow plus). The identical experiment was performed with 30 min preincubation of cells at 4°C followed by 4 h incubation with fluoresceinated peptides
at 4°C to assess temperature dependence of fluorescent labeling.
2.4.12 Serum Stability
The in vitro serum stability assay followed the procedure of previous literature
[13]. Standard solution of PDI-linear (FITC-ßAla-LTFEHYWAQLTS-NH 2 ), PDI-1b
(FITC-(cyclo-5,9)-[ßAla-LTFCHYWS5(2-Me) (R) QLTS]-NH 2 ), and PDI-2b (FITC(cyclo-5,9)-[ßAla-LTFCHYWS 5 (2-Ph) (R) QLTS]-NH 2 ) was prepared in water. Each