Guanidino- and Urea-Modified Dendrimers as Potent Solubilizers of Misfolded Prion Protein Aggregates under Non-cytotoxic Conditions. Dependence on Dendrimer Generation and Surface Charge

Abstract

Amino-terminated dendrimers are well-defined synthetic hyperbranched polymers and have previously been shown to destabilize aggregates of the misfolded, pathogenic, and partially protease-resistant form of the prion protein (PrPSc), transforming it into a partially dissociated, protease-sensitive form with strongly reduced infectivity. The mechanism behind this is not known, but a low pH, creating multiple positively charged primary amines on the dendrimer surface, increases the efficiency of the reaction. In the present study, surface amines of the dendrimers were modified to yield either guanidino surface groups (being positively charged at neutral pH) or urea groups (uncharged). The ability of several generations of modified dendrimers and unmodified amino-terminated dendrimers to deplete PrPSc from persistently PrPSc-infected cells in culture (SMB cells) was studied. It was found that destabilization correlated with both the generation number of the dendrimer, with higher generations being more efficient, and the charge density of the surface groups. Urea-decorated dendrimers having an uncharged surface were less efficient than positively charged unmodified- (amino) and guanidino-modified dendrimers. The most efficient dendrimers (generation 4 (G4) and G5-unmodified and guanidino dendrimers) cleared PrPSc completely by incubation for 4 days at less than 50 nM. In contrast to both unmodified and guanidine-modified dendrimers, the uncharged urea dendrimers showed much lower cytotoxicity toward noninfected SMB cells. Therapeutic uses of modified dendrimers are indicated by the low concentrations of dendrimers needed.

Influence of Surface Functionality of Poly(propylene imine) Dendrimers on Protease Resistance and Propagation of the Scrapie Prion Protein

“…Treatment with mPPI-g5 did not result in changed solubility of PrP C or PrP Sc , but reduced the levels of Numerous studies relate the anti-prion activity of polyamino and phosphorous dendrimers to the generation-dependent density of positively charged surface groups 27,[38][39] 49 . The surface-structure of mPPI is predicted to present a "dense-shell dendrimer" with closely organized maltose units 57 .…”

“…The various dendrimer compounds for which an anti-prion activity has been demonstrated includes the branched polyamines poly(amido amine) (PAMAM), poly(propylene imine) (PPI), and poly(ethylene imine) (PEI) [27][28] and phosphorous 38 dendrimers. Their anti-prion activity in reducing PrP Sc in ScN2a cells is dose-and time-dependent, increases with generation number (size), and is considered to depend on a high density of cationic groups on the surface 27,[38][39] .…”

Dendritic Guanidines as Efficient Analogues of Cell Penetrating Peptides

“…The pyrazole carboxamidine represents an efficient alternative to increase the reactivity of the coupling and was used to functionalize polyamide [29,86], PPI [87,88,89,90,91], melamine [92] and polyether based dendrimers [93]. In the case of polyester based dendrimers [94], pyrazole carboxamidine 2 was used to create a guanidine functionalized precursor with a carboxylic acid moiety 4 that was then coupled to the polyester dendrimer by using carbodiimide chemistry (Figure 4).…”

“…The latter were also hemolytic with a relative hemolysis of 50% (after 24 h) below 10 μg・mL -1 . A 5th generation PPI dendrimer bearing 64 guanidine moieties was significantly more toxic (10 μM for 50% cell viability after 6 days) than the PPI with primary amine groups (40 μM for 50% cell viability after 6 days) [91]. Guanidine functionalized PPI dendrimers showed marked dose dependent cytotoxic effects as well as generation effects meaning that the number of guanidine groups was correlated to the toxicity [86,87].…”

Complex Polyamines: Unique Prion Disaggregating Compounds

“…Given the extreme chemical and physical resistance of infectious prions to inactivation, accomplishing the latter task without disrupting normal cellular physiology would appear daunting. However, in vitro studies with a number of different complex polyamines suggest that this group of chemical compounds might be able to inactivate PrP Sc molecules though unique interactions that do not compromise cell viability [9-14]. The activity, mechanism, and therapeutic potential of these compounds is the subject of this review.…”