Discovery of a Minimally Charged Cell-Penetrating Peptide.

Abstract

Cell-penetrating peptides (CPPs) are powerful tools for delivering membrane-impermeable biomolecules into eukaryotic cells, with broad applications ranging from therapeutics to biopesticides. However, conventional linear CPPs typically require a high density of positive charges (at least +6) to function, often resulting in dose-limiting toxicity and off-target effects. Reducing this charge without sacrificing delivery efficiency remains a significant challenge. In this study, we performed a structure-activity relationship (SAR) analysis and medicinal chemistry optimization of the bismuth-mediated bicyclic CPP, BCP16. This campaign led to the discovery of BCP16e, a potent analog that carries only a +2 charge at physiological pH. Compared to its parent molecule, BCP16e exhibits significantly higher cytosolic entry efficiency, similar proteolytic stability, and a superior safety profile. Our findings demonstrate that high cationic charge is not a prerequisite for efficient translocation, providing a framework for the design of minimally charged, high-efficiency vehicles for intracellular delivery.