Isothiocyanate groups of 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS) inhibit cell penetration of octa‐arginine (R8)–fused peptides.

Delivering biomolecules, such as antibodies, proteins, and peptides, to the cytosol is an important and challenging aspect of drug development and chemical biology. Polyarginine—a well‐known cell‐penetrating peptide (CPP)—is capable of exploiting its positive charge and guanidium groups to carry a fused cargo into the cytosol. However, the precise mechanism by which this occurs remains ambiguous. In the present study, we established a new method of quantitatively assessing cell penetration. The method involves inducing cell death by using a polyarginine (R8) to deliver a peptide—ie, mitochondrial targeting domain (MTD)—to the cytosol. We found that 4,4′‐diisothiocyanatostilbene‐2,2′‐di‐sulfonate (DIDS)—an anion channel blocker—inhibited the ability of octa‐arginine (R8)–fused MTD to penetrate cells. Other anion channel blockers did not inhibit the penetration of peptides fused with R8. Comparison of DIDS with other structurally similar chemicals revealed that the isothiocyanate group of DIDS may be primarily responsible for the inhibitory effect than its stilbene di‐sulfonate backbone. These results imply that the inhibitory effect of DIDS may not be derived from the interaction between stilbene di‐sulfonate and the anion channels, but from the interaction between the isothiocyanate groups and the cell membrane. Our new MTD method enables the quantitative assessment of cell penetration. Moreover, further studies on the inhibition of CPPs by DIDS may help clarify the mechanism by which penetration occurs and facilitate the design of new penetrative biomolecules. [ABSTRACT FROM AUTHOR]