In vitro Pharmacology and permeability assays
The predicted LogS and LogD at different pH values for each structure
were calculated using tools available at Chemicalize.com. The PDE-5
inhibitory activity of sildenafil and malonyl-sildenafil was measuredin vitro using a commercially available kit according to the
manufacturer instructions (BPS Bioscience, San Diego, CA). The assay was
reproduced in three independent experiments with triplicate wells in
each replicate. IC50 values were calculated using curve
fitting functions in GraphPad Prism 9 (San Diego, CA).
The cell-based permeability assays were performed using LS-174T colon
cancer cells obtained from the American Type Culture Collection. Cells
were treated with 1 µM linaclotide together with different PDE5i
concentrations for 4h, followed by processing of cell extracts for
cellular cGMP measurement using cGMP ELISA kits according to the
manufacturer instructions (Cayman Chemical). To assess activation of
cGMP signaling by immunoblotting, we used LS174T cells with inducible
PKG2 expression that were described recently (Hou et al., 2022).