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).