A Supramolecular Approach toward Bioinspired PAMAM‐Dendronized Fusion Toxins

Abstract

Nature has provided a highly optimized toolbox in bacterial endotoxins with precise functions dictated by their clear structural division. Inspired by this streamlined design, a supramolecular approach capitalizing on the strong biomolecular (streptavidin (SA))-biotin interactions is reported herein to prepare two multipartite fusion constructs, which involves the generation 2.0 (D2) or generation 3.0 (D3) polyamidoamine-dendronized transporter proteins (dendronized streptavidin (D3SA) and dendronized human serum albumin (D2HSA)) non-covalently fused to the C3bot1 enzyme from Clostridium botulinum, a potent and specific Rho-inhibitor. The fusion constructs, D3SA-C3 and D2HSA-C3, represent the first examples of dendronized protein transporters that are fused to the C3 enzyme, and it is successfully demonstrated that the C3 Rho-inhibitor is delivered into the cytosol of mammalian cells as determined from the characteristic C3-mediated changes in cell morphology and confocal microscopy. The design circumvents the low uptake of the C3 enzyme by eukaryotic cells and holds great promise for reprogramming the properties of toxin enzymes using a supramolecular approach to broaden their therapeutic applications.

Boosting Antitumor Drug Efficacy with Chemically Engineered Multidomain Proteins

“…The toxin enzyme provides considerable therapeutic relevance in oncology to prevent endothelial cell migration and vessel formation in vitro and in vivo, thereby regulating capillary formation and tumor angiogenesis. But its application has been significantly hampered by its very low uptake into most cell types, and to date, only non cell‐type selective fusions of C3 enzyme have been reported . To chemically engineer the multiprotein complex, bacterial C3 enzyme has been attached to the SST3‐Avi by exploiting the high‐affinity supramolecular biotin–avidin interaction .…”

Functional protein nanostructures: a chemical toolbox

“…In this manner, the optimal stoichiometric ratio of the PAMAM dendrons and the protein enzymes, cytochrome c or tumor suppressor p53, were mixed with streptavidin at room temperature in phosphate buffer to give the tricomponent heterodimeric protein (Fig. 9d) and dendron-induced intracellular delivery of the protein cargo was successfully demonstrated 51,198. Dynamic light scattering (DLS) measurements supported the formation of the AB-type proteins with narrow size distribution (Fig.…”

“…Weil et al first proposed a combinatorial approach where streptavidin was used as a supramolecular “glue” to fuse synthetic entities such as mono-biotinylated polyamidoamine (PAMAM) dendrons, with protein enzymes to mimic binary protein structures of AB-type bacterial toxins comprising of binding and catalytic domains 51,198. Here, the optimization of the biotinylated synthetic entity required for saturation of the binding pockets of streptavidin allows more precision in the stoichiometric loading of the cationic polyamidoamine (PAMAM) dendrons and the protein enzyme of interest, with high conjugation efficiency due to the strong streptavidin–biotin interactions.…”

“…10). 19,20 In this manner, two-faced “Janus-like” PBs were derived to build up non-covalent heteroprotein nanostructures such as a heterotrimer 19,20,198. Notably, chemically post-modified PBs were applied to confer additional functions to the PNs, which could not be achieved by recombinant engineering such as the utilization of HSA incorporated with positively charged dendrons to enhance their cellular uptake 200…”

“…The thus-prepared heteroprotein dimer still consisted of free binding pockets on avidin, which were used for further conjugation to other (imino)biotinylated molecule of interest, e.g. enzymatic proteins such as β-galactosidase and toxin enzymes 20,198. Fluorescence polarization is influenced by changes in molecular weight/hydrodynamic radius, thus affecting molecular mobility 201.…”