We report N-alkylpyridinium reagents for site-selective dual modification of proteins via a regioselective 1,6-thiol addition reaction. The N-alkylpyridinium derivatives can be syn-thesized in two reaction steps and demonstrate high labelling efficiency and chemose-lectivity towards cysteine residues. This reaction is combined with strain-promoted azide-alkyne click and inverse-electron-demand Diels−Alder reactions to achieve dual functionalization of proteins in a sequential one-pot reaction. A Rho-inhibiting enzyme is functionalized with a cancer cell-targeting peptide and a fluorescent dye for successful in vitro uptake imaging and concomitant inhibition of specific Rho-mediated cellular pathways. The ease of synthesis, fast kinetics, high solubility and regioselectivity make N-alkylpyridinium reagents unique for protein/peptide modification to increase their functional diversities for medical applications.

Metastatic breast carcinoma (BC) cells are prone to spreading in the bone microenvironment, leading to a vicious cycle between local osteoclast-mediated osteolysis and tumor progression. Therefore, the targeted pharmacological down-modulation of BC cell proliferation as well as osteoclast differentiation and hyperactivity might represent a promising treatment option. We developed a multifunctional peptide nanocarrier combining bioactive EPI-X4 peptides and the Rho-inhibiting C3bot enzyme from Clostridium botulinum. C3bot is preferentially internalized into the cytosol of monocytic cells, including osteoclasts, where it inhibits Rho-mediated signal transduction. However, Rho-mediated cellular processes like migration and cell division can also be inhibited in non-monocytic cells if C3bot is delivered into their cytosol by a nanocarrier. To accomplish this, we designed a supramolecular transporter where one molecule of biotinylated C3bot and three biotinylated entities of the human EPI-X4 peptide-derived CXCR4 antagonist JM173 are assembled on avidin as a central platform. This modular transport system (JM173)₃-Avi-C3 down-modulated osteoclast formation and hyperactivity and delivered the therapeutic cargo C3bot successfully into the cytosol of breast cancer cells, where it inhibited Rho.