Source Name: 2022_Bhardwaj

Literature Information

  Peptides List

  Statistics

Literature Title Accurate de novo design of membrane-traversing macrocycles
Doi 10.1016/j.cell.2022.07.019
Research Group
  1. Institute for Protein Design, University of Washington
  2. Department of Medicinal Chemistry, University of Washington
  3. Department of Biochemistry, University of Washington
  4. Institute for Molecular Bioscience, University of Queensland
  5. Department of Chemistry and Chemical Biology and Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute
  6. Departamento de Quίmica Fίsica, Universidad de Valencia
  7. Department of Medicine, University of Washington
  8. Department of Chemistry and Biochemistry, University of California-Los Angeles
  9. Takeda Pharmaceuticals Inc.
Data Number 136
Minimum Molecular Weight 622.8
Maximum Molecular Weight 1299.7


Assay Information 1

Assay Type PAMPA
Permeability Type logPapp
Membrane Measurement Direction (Exclude PAMPA) -
Assay Detail Passive permeability was assayed using standard methods on a Corning BioCoat Pre-coated PAMPA Plate System (Kansy et al., 1998). Starting stock solutions of peptides were prepared by adding 1-2 mg of peptide in 1ml of DMSO solution. Stock solutions were diluted 20X in Phosphate buffered saline (PBS) buffer to create solutions with 5%DMSO. 300 uL of peptide solution was added to the donor well and 250 uL of 5%DMSO 1X PBS was added to the acceptor well. Donor and acceptor plates were incubated together for 16-20 hours and transferred to 96-well plates at the end of incubation for measuring concentrations of peptide in starting solution, donor wells, and acceptor wells using an RP-HPLC and mass spectrometry on Agilent 6230 LC/TOF. Samples were separated on a 20%/min gradient of Solvent A (water, 0.1% formic acid) and Solvent B (acetonitrile, 0.1% formic acid) ran using Acquity UPLC BEH C18 1.7 mm column. The area under the curve for the peaks matching the peptide mass was calculated, and peptide concentrations were calculated by fitting sample peak areas to a calibration curve of an 8 point two-fold serial dilution series from the starting donor solution. Propranolol and Cyclosporine were used as positive controls during the PAMPA. Apparent permeability (Papp) were calculated as follows:Papp = -ln(1 - CA/Ce)/A * (1/VD + 1/VA) * t; Ce = (CD * VD + CA * VA)/(VD + VA); t = incubation time (s), CA = compound concentration in the acceptor well at t, CD = compound concentration in the donor well at t, Ce = compound at equilibrium concentration, VA = acceptor well volume, VD = donor well volume, A = filter area.


Assay Information 2

Assay Type Caco2
Permeability Type logPapp
Membrane Measurement Direction (Exclude PAMPA) Apical to Basolateral (AB)
Assay Detail The permeability of designed peptides was evaluated using an accelerated 6-day Caco-2 permeability assay as described previously (Sevin et al., 2013). In brief, cells were seeded into the 12-well Transwell plates with 0.4 mm polyester membrane inserts (Corning, Merck) to give 4.0 3 105 cells/well. The complete assay medium contained Dulbecco’s modified Eagle medium supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) nonessential amino acids, and 0.4 mg/mL puromycin. The medium in plates was renewed every second day. The inserts received 0.75 mL of assay medium in the upper compartment (apical chamber) and 1.5 mL in the bottom compartment (basolateral chamber) individually. After 6-day incubation or until the cells formed monolayers, the medium in the apical and basolateral chamber was replaced with peptide solutions at 50 mM or Hanks’ Balanced Salt Solution (HBSS), separately. The plates were incubated for 90 min at 37°C on an orbital shaker (60 rpm). The peptide concentration of solutions in the apical and basolateral compartments was subsequently quantified using LC-MS (Qstar Elite, AB Sciex Australia) to evaluate the transmembrane flux of peptides in the apical-to-basolateral direction. Membrane integrity of the Caco-2 cell monolayer was validated using transepithelial electrical resistance (TEER) measurements before and after the assays. Atenolol and quinidine were included as negative and positive controls in all assays.