| Assay Detail |
PAMPA was conducted using a 96-well donor plate with 0.45 µm hydrophobic Immobilon-Pmembrane supports (Millipore), and a 96-well Teflon acceptor plate. 160 µM (theoretical concentration) solutions of the sub-libraries (4 µM for each compound) for Library 1 and 180 µM (theoretical concentration) solutions of the sub-libraries (5 µM for each compound) for Library 2 were prepared in 5% DMSO/PBS buffer. 5 µL of a 1% (w/v) solution of lecithin inn-dodecane was carefully applied to the membrane supports in the wells of the donor plate. Without allowing this solution to evaporate, 150 µL of test compounds solutions were added to the donor wells. Each sub-library was prepared in triplicate. The acceptor plate was prepared by adding 300 µL of 5% DMSO/PBS to each well. The donor plate was then placed on top of the acceptor plate so that the artificial membrane was in contact with the buffer solution below. A lid was put on the donor well, and the system was placed in a chamber with wet paper towels to prevent evaporation for approximately 14h (the actual elapsed time was recorded).
Each 50 µL of donor well solution, acceptor well solution, and initial test sub-library solution were mixed with 50 µL of the internal standard (10 µM Tyr(tBu) in MeOH). Then, the relative concentrations were analyzed by LCMS analyses performed on HPLC (UltiMate 3000, Dionex) with attached mass spectrometer (Orbitrap Velos Pro, Thermo Scientific). The 10 µL of sampleswere injected on a 1.9 µm C18 column (Hypersil GOLD 30x2.1 mm, Thermo Scientific) then eluted by the mixture of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) with a 0.6 mL/min flow rate (0-0.5 min, B: 10%, 0.5-3.5 min, B: 10%-100% linear gradient, 3.5-5.0 min, B: 100%, 5.0-6.5 min, B: 10%). PAMPA parameters were obtained byautomatic analysis of the LCMS data using a python program developed in-house. The program extracted single-ion chromatograms for each mass of interest and found the bounds of the peak at the appropriate retention time for that compound. The ion counts between the peak bounds were then summed and used to calculate the PAMPA parameters. In the case of unknown retention time and multiple peaks in the reference spectrum, the highest peak in the reference spectrum was chosen and its retention time was used for peak-finding in the donor and acceptor wells. All peak-picking and integration bounds were reviewed visually and corrected when necessary. In cases where a contaminant peak overlapped the correct peak, integration bounds were adjusted to split the peaks vertically. The ion counts in for each compound were corrected using the ion count of an internal standard to compensate for instrument injection error. PAMPA permeability parameters were calculated by the following formulae: Cequilbrium = (CD*VD + CA*VA)/(VD+VA), %T = CA/Cequilbrium * 100, Papp = (-ln(1-CA/Cequilbrium))/((1/VA + 1/VD) * Area * Time), %recovery = (CD*VD + CA+VA)/(C0*VD) * 100. CD: relative concentration in donor well, CA: relative concentration in acceptor well, C0: relative concentration of initial test solution added to donor well, VD: volume of donor well (0.15 cm^3), VA: volume of acceptor well (0.30 cm^3), Area: membrane area (0.24 cm^2), Time: actual elapsed time in second (51480 sec for side chain library, 51120 sec for diverse scaffold library).
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