Investigation of the Enhancement of Cellular Uptake of PKA Inhibitors via β-Penetratin: A Modified Cell-Penetrating Peptide

Abstract

This study explored whether a modified cell-penetrating peptide, β-penetratin could improve the delivery of the PKA inhibitor PKI-(6-22) into HEK293T cells. The aim was to determine if better delivery would reduce forskolin-induced CREB phosphorylation which is a direct read-out of PKA activity. Confocal microscopy and flow cytometry showed that forskolin activated cAMP/PKA/CREB pathway as expected but none of the penetratin-PKI treatments were able to block this response. SDS-PAGE revealed that the conjugation chemistry used to link penetratin and PKI did not produce a clean, functional product but instead formed unwanted peptide oligomers. These chemical issues likely prevented the inhibitor from entering the cytosol or remaining active inside the cells. Although the delivery did not succeed, the study identifies key problems in the conjugation and stability of CPP-PKI conjugates providing important guidance for improving future peptide delivery systems targeting PKA signaling.