Source Name: 2024_Faris
Literature Information
| Literature Title | Membrane Permeability in a Large Macrocyclic Peptide Driven by a Saddle-Shaped Conformation |
| Doi | 10.1021/jacs.3c10949 |
| Research Group |
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| Data Number | 234 |
| Minimum Molecular Weight | 966.3 |
| Maximum Molecular Weight | 1076.5 |
Assay Information 1
| Assay Type | PAMPA |
| Permeability Type | logPe |
| Membrane Measurement Direction (Exclude PAMPA) | - |
| Assay Detail |
A modified PAMPA was used for compound permeability measurements and was performed by Pharmaron. Briefly, alkyne stock solution (1 mM in DMSO) was diluted (50 μM final in PBS, pH 7.4). The lipid solution (1.8% w/v egg lecithin in dodecane, 5 µL spotting volume) was added to each acceptor well of the multiscreen-IP filter plate. 300 μL PBS was added to all wells of the acceptor plate and diluted alkyne stock (300 µL) was added to the wells of the donor plate in triplicate. The plate was assembled and incubated for 16 h at 37 °C. An aliquot of donor well sample (2.5 µL) was diluted in PBS (47.5 µL) and an aliquot of the acceptor well (50 μL) was transferred to a 96-well analysis plate. Internal standard (100 nM alprazolam, 200 nM caffeine, 200 nM diclofenac in 100% MeOH) was added to all samples. Samples were vortexed and centrifuged (20 min, 3,220 × g), then analyzed by LC-MS/MS.
The effective permeability (Pe) was calculated as: LogPe = Log{Cx[-Ln(1-drug_acceptor/drug_equilibrium)]}, C = VDxVA/[(VD+VA)xtxA] Where VD is the donor compartment volume (0.3 mL), VA is the acceptor compartment volume (0.3 mL), A is the filter area (0.24 cm^2), and t is the time (16 h). |
Assay Information 2
| Assay Type | MDCK |
| Permeability Type | logPapp |
| Membrane Measurement Direction (Exclude PAMPA) | N.D |
| Assay Detail |
The gMDCKI cell line (overexpressed cell line of Madin-Darby Canine Kidney which acquired from the NIH) maintained in DMEM medium supplemented with 1% pen-strep, 5 µg/mL Plasmocin, and 10% FBS under standard culture conditions (37 ºC, 95% humidity, and 5% CO2), were seeded (0.75 x 105 cells) on the HTS Transwell-96 permeable support (Corning) to form a confluent monolayer for two days. All experimental steps were fully automated on Hamilton Vantage. Compound stock solution (1 mM in DMSO) was diluted (10 μM final in HBSS, pH 7.4) with Lucifer Yellow as leakage indicator (20 μM). After washing with HBSS buffer, the solution of test compounds was added to the apical side of the monolayers. The plate was incubated for 3 h at 37 ºC. T0 samples (10 µL of the 10 µM compounds diluted solution with LY) were transferred to a 96-well plate, and then 40 µL of HBSS was added. After 3 hours, 50 μL basolateral samples in the receiving plate were transferred to a fresh 96-well plate and added 100 μL of IS solution (Propranolol). LY were read by plate reader and the samples were analyzed by LC/MS/MS.
The apparent permeability coefficient (Papp) is calculated from the permeation rate and compound concentration at t=0h and t=3h. Where: Papp = (dQ/dt)x(1/(A*C0)), dQ/dt: amount of product present in the basal (A-B) or apical (B-A) compartment as a function of time (nmol/s). A: area of transwell (cm^2). C0: initial concentration of product applied in the apical (A-B) or basal (B-A) compartment (nmol/ml). |