Viologen-Phosphorus Dendrimers Inhibit α-Synuclein Fibrillation

Article pubs.acs.org/molecularpharmaceutics

Viologen-Phosphorus Dendrimers Inhibit α‑Synuclein Fibrillation Katarzyna Milowska,*,† Justyna Grochowina,† Nadia Katir,‡,§ Abdelkrim El Kadib,§ Jean-Pierre Majoral,‡ Maria Bryszewska,† and Teresa Gabryelak† †

Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 141/143 Pomorska Street, 90-236 Lodz, Poland ‡ Laboratoire de Chimie de Coordination CNRS, 205 route de Narbonne, 31077, Toulouse, France § Institute of Nanomaterials and Nanotechnology (iNANOTECH)-MAScIR (Moroccan Foundation for Advanced Science, Innovation and Research), ENSET, Avenue de I’Armée Royale, Madinat El Irfane, 10100 Rabat, Morocco ABSTRACT: Inhibition of α-synuclein (ASN) fibril formation is a potential therapeutic strategy in Parkinson’s disease and other synucleinopathies. The aim of this study was to examine the role of viologen-phosphorus dendrimers in the αsynuclein fibrillation process and to assess the structural changes in α-synuclein under the influence of dendrimers. ASN interactions with phosphonate and pegylated surfacereactive viologen-phosphorus dendrimers were examined by measuring the zeta potential, which allowed determining the number of dendrimer molecules that bind to the ASN molecule. The fibrillation kinetics and the structural changes were examined using ThT fluorescence and CD spectroscopy. Depending on the concentration of the used dendrimer and the nature of the reactive groups located on the surface, ASN fibrillation kinetics can be significantly reduced, and even, in the specific case of phosphonate dendrimers, the fibrillation can be totally inhibited at low concentrations. The presented results indicate that viologen-phosphorus dendrimers are able to inhibit ASN fibril formation and may be used as fibrillar regulating agents in neurodegenerative disorders. KEYWORDS: α-synuclein, viologen-phosphorus dendrimers, fibrillation, circular dichroism

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INTRODUCTION Proteins, as one of the main components of cells, perform many important functions in the body, and their properties depend on their spatial structure. It has been shown that particles with a disordered spatial structure are characterized by the presence of the β structure, and consequently proteins have a tendency to aggregate.1 The tendency of proteins to form insoluble fiber is an important problem in biotechnology and the pharmaceutical industry, because aggregated proteins do not possess the same biological properties as proteins in the native state. Aggregated proteins may be immunogenic, and may even show a strong toxicity effect.2 Inhibition of the accumulation of insoluble protein deposits can have a potential therapeutic application. Earlier studies have demonstrated that polyamidoamine (PAMAM) and polypropylenoimine (PPI) dendrimers, due to their unique branched structure, are promising candidates for the treatment of neurodegenerative changes. The results showed that dendrimers inhibit the formation of insoluble forms of protein, and also remove the existing ones. This suggests that the mechanism of action is not limited to blocking the monomers from which deposits are formed, but probably the existing aggregates are disrupted.3−6 © 2013 American Chemical Society

One of the most important proteins involved in many neurodegenerative diseases is α-synuclein (ASN). ASN is a presynaptic neuronal protein, whose normal function has not been yet established; however, several studies suggest that ASN plays a role in the regulation of the synaptic function, dopaminergic system, neuronal plasticity, and vesicular transport.7 Misfolded protein and the accumulation of its aggregates is a key event in the pathological cascades of several neurodegenerative disorders, e.g., it is one of the basic components of Lewy bodies, whose presence has been established in degenerated neurons.8 A better understanding of the biological role played by α-synuclein is a major challenge for researchers. In order to prevent aggregation of ASN, it is important to know which compound will effectively prevent the degradation of this protein. Dendrimers, thanks to their properties, are potential candidates for the inhibition of this process. The positive effect of cationic polyamidoamine (PAMAM) and phosphorus dendrimers on the process of α-synuclein aggregation has been already demonstrated.9−11 Received: Revised: Accepted: Published: 1131

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Figure 1. Viologen-phosphorus dendrimer structures.

chemical structures of the generation 0 dendrimers are shown in Figure 1.

Specifically, PAMAM G3, G4 and G5 dendrimers inhibit the α-synuclein fibrillation process by preventing the formation of the β structure in its molecule. The inhibition efficiency increases with both the dendrimer concentration and the generation number.10 The mechanism of dendrimers action is not limited only to inhibiting the growth of fibrils. These macromolecules are also involved in the breaking and degradation of the existing protein aggregates. In addition, the nature of functional peripheral groups plays a pivotal role in the inhibition of the α-synuclein aggregation process. For instance, it has been demonstrated that PAMAM G3.5 dendrimers decorated with carboxyl groups on the surface do not allow the inhibition of α-synuclein aggregation.9 Also, phosphorus containing dendrimers may be potential inhibitors of α-synuclein fibrillation, however, their effectiveness depends on the used concentration and the size of dendrimers. These studies showed that phosphorus dendrimers at lower concentrations inhibited the aggregation of α-synuclein. The higher concentrations did not inhibit the fibril formation process. The phosphorus dendrimer G3 was a better inhibitor of the aggregation process than G4. It is suggested that these results are related to the large size of the particles of dendrimers.11 In this study, as potential factors in preventing fibrillation of ASN in vitro, we selected two viologen-phosphorus containing dendrimers, new, highly branched polymers, whose biological properties are still insufficiently known. These types of compounds exhibit antimicrobial properties, and their behavior depends on the number of viologen units, the size and the nature of the surface groups.12 The dendrimers containing viologen demonstrated antiviral activity against the human immunodeficiency virus (HIV) and, to a lesser extent, against other viruses (herpes simplex virus, Reovirus and respiratory syncytial viruses).13 In the present work, we investigated whether polycationic viologen-phosphorus dendrimers (two positive charges per viologen unit) form complexes with ASN and can inhibit ASN fibrillation. These compounds are characterized by the presence of a hexafunctionalized core (P3N3)(NCH3N)6, viologen linkages as branches and either phosphonate groups or polyethylene glycol (PEG) groups on their surface. The

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EXPERIMENTAL SECTION

Materials. Viologen-phosphorus containing dendrimers were synthesized according to the protocol previously described.12 Human ASN was purchased from Sigma-Aldrich (USA). For all experiments, ASN and dendrimers were dissolved in phosphate-buffered saline (1.9 mM NaH2PO4, 8.1 mM Na2HPO4, pH = 7.4). All the other chemicals were of analytical grade. All solutions were made with water purified by the Milli-Q system. Zeta Potential. The measurements of the zeta potential (electrokinetic potential) were performed with Zetasizer Nano ZS from Malvern, which uses electrophoresis and LDV (laser Doppler velocimetry) techniques. Applying a combination of these two techniques allowed measuring the electrophoretic mobility of the molecules in the solution. The zeta potential value was calculated directly from the Helmholtz−Smoluchowski equation using the Malvern software.14 The measurements were performed at 37 °C with three repetitions. Increasing concentrations of the dendrimer in the range 0.1−10 μM were added to α-synuclein of a concentration of 0.2 μM, and zeta potential was measured. Thioflavin T Fluorescence Measurements. The process of fibrillation was monitored using dye thioflavin T (ThT), whose fluorescence depends on the presence of aggregates. The samples of ASN alone and ASN in the presence of viologen-phosphorus dendrimers were incubated at 37 °C for 0, 8, 24, 32, 48, and 72 h. The concentration of ASN was 2 μM, and the concentrations of viologen-phosphorus dendrimers were 1, 2, 3, and 4 μM. After incubation, ThT at a final concentration of 15 μM was added to the samples with the protein and the protein with dendrimers. Fluorescence measurements were performed at 37 °C using a Perkin-Elmer LS-50B fluorimeter with the excitation at 450 nm, and the emission at 490 nm. Before examining the fluorescence with ThT, it was made sure that viologen-phosphorus dendrimers did not bind ThT and did not emit fluorescence. 1132

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dendrimers changed from −21 mV to −6 mV for vpd-1 and −3 mV for vpd-2 (Figures 2A and 2B). The analysis of possible changes of the zeta potential as a function of the molar ratio can be approximated by the number of dendrimer molecules that can attach to one molecule of α-synuclein. The number of binding centers is the point (X,O) corresponding to the point of intersection of two straight lines tangent to the curve of the zeta potential as a function of the molar ratio. The calculated n is 1.5 for vpd-1 and 1 for vpd-2. Thioflavin T Fluorescence. Fibril formation was monitored using the fluorescent dye thioflavin T (ThT). ThT is a highly selective dye whose fluorescence depends on the formation of protein fibrils. Figure 3 demonstrates how

Circular Dichroism (CD) Spectroscopy. The CD spectra (195−260 nm) were recorded for ASN in the presence/ absence of dendrimers on a Jasco J-815 CD spectropolarimeter, in 10 mm path length quartz cuvettes, with a wavelength step of 1 nm, a response time of 4 s and a scan rate of 20 nm/min, thermostatted at 37 °C. Each spectrum was the average of three repetitions. Viologen-phosphorus dendrimers alone at the concentration used in the experiments did not produce any discernible features in their CD spectra and were used as blanks for ASN-dendrimer complex spectra. The changes in the secondary structure of ASN in the presence and absence of dendrimers were tested immediately and after 24, 48, and 72 h of incubation at 37 °C. In these experiments, we used ASN at a concentration of 2 μM and dendrimers at the concentrations of 1, 2, 3, and 4 μM. Statistical Analysis. The results of the fluorescent study and zeta potential are presented as mean ± SD from six individual experiments. Each experiment was repeated three times. Statistical evaluation of the difference between the control and the treated group was performed using Student’s t test. P < 0.05 and below was accepted as statistically significant.

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RESULTS

Zeta Potential. The changes in the zeta potential of ASN upon addition of viologen-phosphorus dendrimers in the molar ratio range 0−25 for vpd/ASN were studied. The results are presented in Figure 2. The zeta potential of α-synuclein has a value of −21 mV. The addition of viologen-phosphorus dendrimers led to significant changes in the values of the zeta potential but did not change the sign of the potential, it was still negative. The values of the zeta potential after adding

Figure 3. Thioflavin T fluorescence during the fibrillation process of αsynuclein in the presence of dendrimers (A) vpd-1, (B) vpd-2.

viologen-phosphorus dendrimers affect α-synuclein aggregation. A control curve (ASN without dendrimer) shows a typical fibrillation process, where fibrils were systematically created, and a plateau was obtained after 48 h of incubation. After the incubation of ASN with viologen-phosphorus dendrimer vpd-1 at concentrations 1 and 2 μM, the fibrillation process was completely inhibited. For the two higher concentrations of vpd-1 (3 and 4 μM) and all concentrations of dendrimer vpd-2 a slight increase in ThT fluorescence intensities starting from 32 h of incubation was observed. However, independently of the elongation of the incubation time the fluorescence intensity was at the same level. Circular Dichroism (CD) Spectroscopy. To correlate the amount of formed fibrils, which were observed in ThT measurements, with the changes in a secondary structure of ASN, CD experiments were carried out. ASN CD spectra are presented in Figure 4. α-Synuclein is a natively unfolded protein and exhibits a random coil structure under physiological

Figure 2. α-Synuclein zeta potential in the presence of viologenphosphorus dendrimers (A) vpd-1, (B) vpd-2. 1133

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dendrimer. In the case of the second dendrimer (vpd-2), minor changes were observed at the minimum, but these changes did not lead to the conversion into the β form. These results indicated that the structure of ASN was still disordered. Summarizing, after 48 h of incubation, the CD spectra of ASN in the presence of dendrimers were very similar to the spectrum of native ASN, indicating that the used dendrimers inhibited α-synuclein fibrillation. The results after 24 and 72 h of incubation are not presented because there are no significant differences compared to the data obtained after 48 h.

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DISCUSSION Protein aggregates formed in the central nervous system contribute to many neurodegenerative diseases.15,16 Fibril formation and the presence in the brain of deposits of pathological protein are characteristic of neurodegenerative diseases such as Parkinson’s, Alzheimer’s or dementia with Lewy bodies.17 Nowadays many research groups are looking for factors or agents that could prevent or inhibit the aggregation process. Unique properties of dendrimers and the possibility of their clinical use have led to studies on the interaction between dendrimers and cellular components, mainly proteins. Viologen-phosphorus dendrimers, which were used in this work, contain in their framework viologen units (derivative of 4,4′bipyridine salt) and phosphonate groups or polyethylene glycol (PEG) groups on their surface. The presence of phosphonate groups on the surface of one of these dendrimers is very important due to their antiinflammatory properties,18 and NK cell amplification,19 etc. The results obtained in earlier research also demonstrated that polycationic phosphorus dendrimers exhibit anti-HIV activity,20 can be used as transporters of drugs and genetic material21,22 and can prevent aggregation of proteins associated with neurodegenerative processes, namely, prion,23 Alzheimer’s and Parkinson’s diseases,3,11,24 whereas the presence of PEG groups on the surface of the second dendrimer may reduce toxicity and increase the solubility in water. It was also demonstrated that viologen dendrimers have antiviral properties, especially against HIV.13 Interactions between viologen-phosphorus dendrimers and α-synuclein have been studied by measuring the zeta potential. The zeta potential characterizes the electric double layer, which is formed on the molecule−liquid phase boundary. Zeta potential value can be related to the dispersion stability or the ability to maintain the particles in a colloidal dispersion. Unstable dispersions undergo sedimentation or coagulation processes.25 The measurement of the zeta potential also allows determining the number of dendrimer molecules bound to protein and the potential of this complex. As shown in Figure 2, the addition of the dendrimer to α-synuclein resulted in changes in the zeta potential of approximately −21 mV to −6 mV for vpd-1 and −3 mV for vpd-2. Distribution of the charge in α-synuclein is dependent on the solution pH. The net charge is −9 at the neutral pH, but it is different for different regions.26 The used dendrimers have a positive charge (+12), but the viologen groups, which are positively charged, are located inside the molecule. This may explain why the zeta potential of the complex does not reach positive values. Analysis of the results allowed determining the approximate number of dendrimer molecules that can attach to one molecule of α-synuclein. α-Synuclein combined with vpd-1

Figure 4. CD spectra of α-synuclein depending on the incubation time.

conditions. The minimum was observed at 200 nm. After the incubation, we observed a change in the ASN spectrum shape, which indicated changes in its secondary structure. After 48 h of incubation, a positive signal was obtained in the range 195−205 nm in the CD spectrum, which may indicate the conversion of the disordered structure into the β form. CD spectra of α-synuclein alone and α-synuclein incubated with dendrimers for 48 h are shown in Figures 5A and 5B. In the case of vpd-1 dendrimer a slight decrease in negative signal intensity (from −10 to −8) occurred after 48 h of incubation at concentrations of 3 and 4 μM. However, no significant changes in the shape of α-synuclein spectra were observed, suggesting that the structural changes in α-synuclein were inhibited by the

Figure 5. CD spectra of α-synuclein alone and in the presence of dendrimers (A) vpd-1, (B) vpd-2, after 48 h of incubation. The dendrimers were added to α-synuclein at time 0 h. 1134

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Table 1. The Efficiency of Different Types of Dendrimers towards Inhibition of the Fibrillation of α-Synucleina dendrimers PAMAM

phosphorus (pd)

viologen-phosphorus (vpd)

charge

ASN/dendrimer molar ratio

G3.5

MW [Da] 12 928

−COO−

nd groups

−64

G4

14 214

−NH2

+64

G3

16 280

−N+HEt2

+48

G4

33 702

−N+HEt2

+96

G0

3 789

−P(O)(OEt)2

+12

G0

14 421

PEG (M = 2000 Da)

+12

1:1 1:2 1:1 1:2 1:0.1 1:0.5 1:1 1:2 1:0.1 1:0.5 1:1 1:2 1:0.5 1:1 1:1.5 1:2 1:0.5 1:1 1:1.5 1:2

% of inhibition 5.3 3.0 27.2 30.3 63.9 59.7 7.8 6.0 48.5 36.3 3.2 1.2 90.7 86.7 71.8 73.7 73.9 73.0 74.0 71.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4.3 2.8 2.9 3.2 3.0 2.5 3.4 3.0 3.3 2.7 2.7 0.7 3.4 1.9 1.3 1.2 2.1 1.7 2.5 1.9

ThT fluorescence for ASN taken as 100% fibrillation. The percentage of fibrillation was calculated using the following formula: % of inhibition = 100% − (FASN+D/FASN) × 100%, where FASN+D is fluorescence of ASN in the presence of dendrimer and FASN+D is fluorescence of ASN. a

growth phase, which corresponds to fibril elongation, and (iii) the final phase, which corresponds to amount of formed fibrils.27 Two main types of inhibitors of the aggregation process can be distinguished: kinetic and thermodynamic. These types of inhibitors can be identified by analyzing the shape of kinetics curves compared to a control curve. Kinetic inhibitors are characterized by differences in lag time, but the final amount of fibrils is unchanged. In the case of thermodynamic inhibitors, the final amount of fibrils is reduced, but there is no significant effect on the elongation phase.28 As evidenced by the fibrillation kinetics curves, α-synuclein incubation at 37 °C contributes to the spontaneous fibril formation. Depending on the concentration of the dendrimer and its groups on the surface, the process can be slightly or even totally inhibited. A more effective inhibitor is the vpd-1 dendrimer allowing total inhibition under the two lower used concentrations, whereas vpd-2 dendrimer and vpd-1 at higher concentrations did not completely inhibit this process but largely decreased the amount of formed fibrils. These results indicate that viologen-phosphorus dendrimers are thermodynamic inhibitors, because the final number of fibrils is significantly or completely reduced. A similar effect occurred for phosphorus dendrimers.11 Several preliminary observations can be formulated corresponding to the efficiency of different types of dendrimers toward inhibition of the fibrillation of ASN (Table 1). The percent of inhibition is different for different types of dendrimers. The most effective is viologen-phosphorus dendrimer. Inhibition necessitates the presence of positively charged dendrimers, but the number of positive charges for a given type of dendrimer does not dramatically influence the activity of these dendrimers since viologen dendrimers (vpd-1 and -2, 12 positive charges) inhibit ASN fibrillation as PAMAM (64 positive charges) or phosphorus dendrimers (48 or 96 positive charges) do. Negative charges on the surface of dendrimers do not allow the inhibition of the ASN fibrillation. The inhibition also does not depend on the generation number

dendrimer in a 1:1.5 ratio, and one-dendrimer molecule vpd-2 falls on one molecule of α-synuclein. Based on these results, the ratios of dendrimers to α-synuclein which were used in further studies were selected. In addition to the calculated molar ratios, a shortage and excess of dendrimer were used. The influence of viologen-phosphorus dendrimers on the fibrillation of ASN was examined using circular dichroism spectroscopy and the fluorescence methods with ThT. Circular dichroism spectroscopy allows investigating of the secondary structure of proteins and peptides. Alterations in the shape of α-synuclein CD spectra, which show the structural changes, were observed after 24 h incubation at 37 °C. After 48 h of incubation a positive signal indicating the conversion of the α-synuclein disordered structure to the β form was observed. After the addition of viologen-phosphorus dendrimers to αsynuclein and incubation for 48 h, no significant changes in the shape of the spectrum in comparison to nonincubated ASN were noticed. Based on the obtained results, it is concluded that both dendrimers used in all concentrations inhibit the formation of the β structure. The ability to inhibit the fibrillation process of α-synuclein has been demonstrated by the use of cationic PAMAM dendrimers9,10 and phosphorus dendrimers.11 However, the final result of the inhibition of aggregation is dependent on the ratio of phosphorus dendrimers to protein. The results show that dendrimers at lower concentrations are thermodynamic inhibitors, because the final number of fibrils is reduced.11 The work by Klajnert et al.3 demonstrates that phosphorus dendrimers are also able to interfere in the process of peptide Prp 185−208 aggregation, thus reducing the final number of amyloid fibrils. α-Synuclein fibrillation was studied by monitoring the changes in thioflavin T fluorescence intensity, which is dependent on the amount of protein aggregates. The curve of the ASN fibrillation is characterized by the sigmoidal shape, which can be divided into (i) an initiation phase “lag”, which corresponds to the formation of a critical nucleus, (ii) a rapid 1135

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(8) Hong, D. P.; Xiong, W.; Chang, J. Y.; Jiang, Ch. The role of the C-terminus of human α-synuclein: Intra-disulfe bonds between the Cterminus and other regions stabilize non-fibrillar monomeric isomers. FEBS Lett. 2011, 585, 561−566. (9) Milowska, K.; Malachowska, M.; Gabryelak, T. PAMAM G4 dendrimers affect the aggregation of α-synuclein. Int. J. Biol. Macromol. 2011, 48, 742−746. (10) Rekas, A.; Lo, V.; Gadd, G. E.; Cappai, R.; Yun, S. I. PAMAM dendrimers as potential agents against fibrillation of α-synuclein, a parkinson’s disease-related protein. Macromol. Biosci. 2009, 9, 230− 238. (11) Milowska, K.; Gabryelak, T.; Bryszewska, M.; Caminade, A. M.; Majoral, J. P. Phosphorus-containing dendrimers against α-synuclein fibril formation. Int. J. Biol. Macromol. 2012, 50, 1138−1143. (12) (a) Ciepluch, K.; Katir, N.; El Kadib, A.; Felczak, A.; Zawadzka, K.; Weber, M.; Klajnert, B.; Lisowska, K.; Caminade, A.-M.; Bousmina, M.; Bryszewska, M.; Majoral, J. P. Biological properties of new viologen-phosphorus dendrimers. Mol. Pharmaceutics 2012, 9, 448− 457. (b) Katir, N.; Majoral, J. P.; El Kadib, A.; Caminade, A. M.; Bousmina, M. Molecular and macromolecular engineering with viologens as building blocks: rational design of phosphorus-viologen dendritic structures. Eur. J. Org. Chem. 2012, 2, 269−273. (13) Asaftei, S.; De Clercq, E. Viologen” dendrimers as antiviral agents: the effect of charge number and distance. J. Med. Chem. 2010, 53, 3480−3488. (14) Sze, A.; Erickson, D.; Ren, L.; Li, D. Zeta-potential measurement using the Smoluchowski equation and the slope of the current−time relationship in electroosmotic flow. J. Colloid Interface Sci. 2003, 261, 402−410. (15) Szwed, A.; Milowska, K. The role of proteins In neurodegenerative diseases. Postepy Hig. Med. Dosw. 2012, 66, 187−195. (16) Klajnert, B.; Bryszewska, M. Affinity of dendrimers for proteins and peptidesbiomedical implication. In Nanotechnological Applications of Novel Polymers; Adeli, M., Ed.; Translation Research Network: Kerela, India, 2009; pp 51−71. (17) Uversky, V. N.; Li, J.; Bower, K.; Fink, A. L. Synergistic effect of pesticides and metals on the fibrillation of alpha-synuclein: implications for Parkinson’s disease. Neurotoxicology 2002, 23, 527− 536. (18) Hayder, M.; Poupot, M.; Baron, M.; Nigon, D.; Turrin, C. O.; Caminade, A. M.; Majoral, J. P.; Eisenberg, R. A.; Fournie, J. J.; Cantagrel, A.; Poupot, R.; Davignon, J. L. Phosphorus-based dendrimer as nanotherapeutics targeting both inflammation and osteoclastogenesis in experimental arthritis. Sci. Transl. Med. 2011, 3, 81ra35. (19) Griffe, L.; Poupot, M.; Marchand, P.; Marava,l, A.; Turrin, C. O.; Rolland, O.; Metivier, P.; Bacquet, G.; Fournie, J. J.; Caminade, A. M.; Poupot, R.; Majoral, J. P. Multiplication of human Natural Killer cells by nanosized phosphonate-capped dendrimers. Angew. Chem., Int. Ed. 2007, 46, 2523−2526. (20) Caminade, A. M.; Turrin, C. O.; Majoral, J. P. Biological properties of phosphorus dendrimers. New J. Chem. 2010, 34, 1512− 1524. (21) Loup, C.; Zanta, M. A.; Caminade, A. M.; Majoral, J. P.; Meunier, B. Preparation of water-soluble cationic phosphoruscontaining dendrimers as DNA transfecting agents. Chem.Eur. J. 1999, 5, 3644−3650. (22) Maksimenko, A. V.; Mandrouguine, V.; Gottikh, M. B.; Bertrand, J. R.; Majoral, J. P.; Malvy, C. Optimisation of dendrimermediated gene transfer by anionic oligomers. J. Gene Med. 2003, 5, 61−71. (23) Solassol, J.; Crozet, C.; Perrier, V.; Leclaire, J.; Beranger, F.; Caminade, A. M.; Meunier, B.; Dormont, D.; Majoral, J. P.; Lehmann, S. Cationic phosphorous-containing dendrimers reduce prion replication both in cell cultures and in mice infected with scrapie. J. Gen. Virol. 2004, 85, 1791−1799. (24) Shcharbin, D.; Dzmitruk, V.; Shakhbazau, A.; Goncharova, N.; Seviaryn, I.; Kosmacheva, S.; Potapnev, M.; Pedziwiatr-Werbicka, E.; Bryszewska, M.; Talabaev, M.; Chernov, A.; Kulchitsky, V.; Caminade,

and the molecular weight since phosphorus dendrimers (G3 and G4) as well as phosphorus-viologen vpd-1 (generation 0) are active species at the lower concentrations used. However, the presence of phosphonate groups on the surface of vdp-1 allows this dendrimer to be more effective against the inhibition of ASN fibrillation (90.7% of inhibition for ratio 1:0.5) than the corresponding vdp-2 dendrimer of generation 0 bearing terminal PEG groups (≈71−74% of inhibition), thus indicating the influence of terminal groups on the properties of dendrimers. There are three possible strategies of inhibiting the formation of fibrils by dendrimers: reduction of protein concentration as a result of binding to dendrimer, blocking the fibril growth and breaking the existing fibrils.4 It is difficult to unequivocally indicate which mechanism dominates. It seems that all of them are likely.

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CONCLUSION In conclusion, the results obtained in this study suggest that low generation viologen-phosphorus dendrimers can be potential inhibitors of ASN fibril formation. Vpd-1, which possesses phosphonate groups on the surface, is a more effective dendrimer. The fact that dendrimers can prevent ASN fibrillation in suspension is therefore important for further research because it may lead to the design of effective pharmacological strategies.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (48 42) 635 43 80. Fax: (48 42) 635 44 74. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The project was supported by the COST Action TD0802, CNRS and MAScIR foundation. REFERENCES

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