TMCnet News

Cis-Glyco-Fused Benzopyran Derivatives as Hit Compounds for the Development of Therapeutic and Diagnostic Tools against Neurodegenerative Diseases [ChemPlusChem]
[August 25, 2014]

Cis-Glyco-Fused Benzopyran Derivatives as Hit Compounds for the Development of Therapeutic and Diagnostic Tools against Neurodegenerative Diseases [ChemPlusChem]


(ChemPlusChem Via Acquire Media NewsEdge) Oligomeric and fibrillar aggregates generated by amyloid-ß (Aß) and prion protein (PrP) peptides are amongst the principal components of amyloid plaques found post mortem in patients suffering from Alzheimer's disease and mammalian prion diseases. Hence these amyloid peptides represent major molecular targets to develop potential drugs and diagnostic tools for the above-mentioned neurodegenerative diseases. Recently, a small library of cis-glyco-fused benzopyran compounds has been synthesized by us, and their ability to recognize and bind Aß peptide oligomers and stain Aß deposits was demonstrated. By exploiting the structural similarity between Aß and PrP aggregates, herein the interaction of these benzopyran molecules with PrP oligomers and their inhibition of the PrP aggregation process that leads to amyloid fibril formation are investigated. Finally, the in vitro staining of PrP fibrils is achieved with a fluorescently labeled cis-glyco-fused benzopyran derivative able to cross a model of the blood-brain barrier.



Keywords : amyloid beta-peptides · molecular recognition · neurodegenerative diseases · NMR spectroscopy · prion protein Introduction The accumulation of misfolded proteins as insoluble, fibrillar aggregates is a common feature of several degenerative dis- eases. The most significant examples include the proteins in- volved in amyloid diseases such as Alzheimer's disease (amy- loid-b (Ab)),[1] the mammalian prion diseases (prion protein (PrP)),[2] Parkinson's disease (a-synuclein),[3] and also type II dia- betes (amylin).[4] In contrast to natively folded proteins, the specific three-di- mensional structure of which depends strongly on their pri- mary structure, amyloidogenic proteins fold independently of their amino acid sequence and enter aggregation pathways leading ultimately to the formation of fibrillar structures.[5] Among the intermediates of these pathways, soluble oligo- mers are believed to be the most toxic and infective species[6] as well as a common element of all amyloid diseases for many of these proteins. In particular they have some common key features, such as reactivity to structural antibodies, the ability to increase membrane permeability, and cytotoxicity to cul- tured neurons, although their primary amino acid sequences are often very different.[7] Despite the inherent difficulties in the study of aggregative processes, some recent studies shed light on the main structural features of these aggregates ; these results, particularly regarding the characterization of amyloid fibril structures investigated by solid-state NMR spec- troscopy, have been reviewed recently by Tycko.[8] Concerning oligomeric structures, they seem to be characterized by some common properties.[7] According to recent data, for example, both Ab1-40 and PrP106-126 (a peptide deduced from the prion protein sequence) form large oligomers containing fibril- like secondary structures and intermolecular contacts, which suggest a small fibril-like segment composition arranged in a micelle-like assembly.[9, 10] The pivotal role played by oligomers in the pathogenesis of amyloid diseases has fueled an ongoing effort towards the identification of novel compounds able to inhibit their toxicity and prevent their further aggregation to give fibrillar struc- tures. Different groups have worked on the development of antibodies targeting against native and misfolded conforma- tions as well as different kinds of aggregates in the case of both Ab and PrP proteins.[11] At the same time, several small molecules, both natural and synthetic compounds, have been identified and notably some of them are active on oligomers formed by diverse amyloid proteins.[12] In recent years, for ex- ample, we have shown that tetracyclines present anti-amyloi- dogenic activity against Ab and PrP, promoting their degrada- tion by proteases, but also inhibiting the aggregation of amy- loidogenic peptides Ab1-40, Ab1-42, PrP106-126, and PrP82- 146. Tetracyclines present anti-amyloidogenic activity also against other amyloidogenic proteins, such as transthyretin, W7FW14F apomyoglobin, huntingtin, amylin, a2-macroglobu- lin, and a-synuclein.[13] The considerable efforts in the design of molecules able to target amyloid oligomers provided several classes of com- pounds,[14] but, until now, the drugs selected for clinical trials have given unsatisfactory results and the need for molecules useful for both the treatment and diagnosis of these diseases is still urgent.

We have recently described a small library of cis-glyco-fused benzopyran compounds able to recognize and bind Ab oligo- mers.[15] Besides the good affinity for Ab aggregates, these molecules exhibit chemical stability and high solubility in phys- iological conditions. In addition, they can be suitably function- alized to modulate their hydro-/lipophilic balance and intro- duce functional modifications to generate new potential diag- nostic/therapeutic tools.[15] In particular, we developed a fluo- rescent derivative able to cross an in vitro model of the blood- brain barrier (BBB) and stain Ab deposits.[16] Moving from the structural similarity between big, soluble Ab and PrP oligomers, we investigated the ability of our com- pounds to recognize and bind PrP oligomers and interfere with their aggregation process.


Results and Discussion The structure of the compounds screened as potential ligands of PrP106-126 are reported in Scheme 1. Compounds 1-6 are characterized by a glyco-fused benzopyran structure contain- ing an aromatic moiety. It is supposed to be important for in- teraction with amyloid aggregates. The molecules comprising this small library differ in the nature of the substituent on the aromatic ring and in the carbon stereochemistry at position 3.

NMR molecular recognition studies The ability of compounds 1-6 to recognize and bind PrP106- 126 oligomers was investigated by NMR spectroscopy. Owing to the significant structural similarity between the oligomers formed by the two peptides, we adopted the same strategy re- cently applied to the identification and characterization of li- gands of Ab peptide oligomers,[16-19] including molecules 1- 6.[15] Our approach was based on the acquisition of transfer NOESY (trNOESY)[20] and saturation transfer difference (STD) NMR[21] experiments to outline the main structural features of the interaction of low-molecular-weight ligands with amyloid oligomers in solution.

Similarly to what has been achieved for Ab peptides, we ob- tained samples enriched in peptide oligomers by dissolving PrP106-126 in deuterated phosphate-buffered saline (PBS), pH 7.4, 25 8C. The oligomeric form of the PrP aggregates pres- ent in these samples was supported by transmission electron microscopy (TEM). The TEM image clearly shows the presence of two families of globulomer structures with a different di- mension. The predominant family shows a distribution with a diameter in the range of 6-12 nm whereas in the second family an annular structure in the range of 20-23 nm was pres- ent (Figure S1 in the Supporting Information).

STD NMR experiments were performed on a ligand/peptide 10:1 ratio dissolved in deuterated PBS, pH 7.4, 5 8C, to exploit the increase in STD effect at low temperature.[22] The mixture was analyzed following irradiation of the sample at ^0.5 ppm to achieve the selective saturation of some aliphatic resonan- ces of PrP oligomers. Generally speaking, the appearance of signals of the test molecule in the STD NMR spectrum is a clear demonstration of the presence of interaction with PrP aggregates. Conversely, the absence of compound resonances in the STD NMR spectrum indicates that the molecule is not a PrP ligand.

As shown in Figure 1, several NMR resonances of mole- cules 1, 4, 5, and 6 appeared in the corresponding STD NMR spectra (spectra B, H, L, and N, respectively) recorded in the presence of PrP106-126, thus supporting the ability of the compound to recognize and bind PrP oligomers. On the other hand, in the case of compounds 2 and 3 the STD NMR spec- trum shows only aromatic resonances with extremely low in- tensity, clearly indicating that the affinity of these compounds for PrP oligomers is essentially negligible (spectra D and F). These data are in substantial agreement with the results ob- tained for the binding to Ab oligomers. The only exception is compound 3, which demonstrated the ability to recognize and bind Ab aggregates, although it was the ligand with the lower affinity among those screened.[15] The binding of the best ligands (compounds 5 and 6)was further confirmed by the acquisition of trNOESY experiments. The change in the sign of the cross-peaks of the test molecule, from positive when PrP is absent to negative in the presence of peptide oligomers, results from an increase of its effective rotational motion correlation time, and supports its binding to a large molecular entity, that is, PrP106-126 aggregates. Fig- ureS2 reports NOESY (FigureS2A) and trNOESY (Figure S2B) spectra obtained for compound 5.

Thus we performed a competitive STD NMR experiment, by mixing all the compounds in the same sample in the presence of PrP oligomers. With this experiment we ranked com- pounds 1-6 according to their affinity for PrP, since STD inten- sity is proportional to the ligand binding affinity for the molec- ular target.[20] This is true in particular for ligands with dissocia- tion constants kD in the millimolar and micromolar range. In fact, the STD NMR experiment works in the fast-exchange regime, which allows identification of ligands with low and moderate affinities, whereas molecules with very high affinity that usually show very small dissociation constants (koff) can give a poor or negligible STD effect.[20] Nevertheless, as demon- strated by Wang and co-workers, competitive STD NMR spec- troscopy can also be exploited for the detection of high-affini- ty ligands, as the presence of a competing high-affinity ligand in the compound mixture can be detected by the disappear- ance or reduction of the STD signals of a low-affinity reference ligand.[23] Thus, generally speaking, this experiment is an ex- tremely useful, efficient, and versatile tool for the screening of a small library of compounds for which the affinity for the target has to be compared.

For each molecule, we measured the STD effect on H6 and the fractional STD effect was calculated as (I0^I)/I0, in which I is the intensity of the monitored signal in the STD NMR spectrum and I0 is the intensity of the same signal in a reference spec- trum (see Figure 2). Compound 6 showed the highest fraction- al STD effect that was set equal to 1 and, therefore, the relative intensities for the other mole- cules were calculated.

Compounds 5 and 6, which present a methyl substituent on the aromatic ring, are the li- gands with the highest affinity, followed by 4, the aromatic ring of which is substituted by an O- methyl group, and by 2, with no aromatic substituent. As already mentioned, the binding to PrP oligomers of compounds 2 and 3, with two polar groups as aro- matic substituents, is negligible. Thus, the experimental evidence strongly highlights the fact that the lower the polarity of the substituents on the aromatic ring, the higher the molecule af- finity for PrP, which supports the importance of their hydrophobic character to allow for a strong interaction with amyloid oligo- mers.

For ligands 1, 4, 5, and 6, STD NMR experiments were per- formed with five different satura- tion times (0.5, 1.2, 2.0, 3.0, 4.0 s) to derive indications about the binding epitopes from the STD buildup (Figures S3-S6). Figure 3 shows the STD NMR spectra acquired on com- pound 5.

Relative STD effects calculated for all the nonoverlapping protons of compounds 1, 4, 5, and 6 are presented in Figure 4, from which the STD NMR-based binding epitopes can be de- duced.

This analysis indicates that the region of the ligands playing a pivotal role in the interaction with PrP oligomers is the aro- matic ring. On the contrary, protons of the saccharide portion exhibit the least intense STD signals, thus suggesting that this portion is not directly involved in the molecular recognition process. This evidence fits with the lack of influence on the binding affinity for the stereochemistry of sugar carbon atoms. In fact, compounds 5 and 6, which differ only in the stereo- chemistry at position 3, show the same affinity (Figure 2).

All these findings are coherent with the results obtained pre- viously with Ab peptide,[15] except for the lack of interaction between compound 3 and PrP oligomers. Hence, the NMR in- teraction studies performed in solution clearly indicate that this small library of compounds ex- hibits the same binding mode as both Ab and PrP oligomers.

Anti-amyloidogenic activity To verify if compound 5, one of the two best ligands identified by NMR interaction studies, was able to interfere with the aggre- gation of PrP106-126 that leads to the formation of mature fibers, a TEM morphological analysis was performed. PrP106- 126 (0.5 mm) was dissolved in PBS buffer in the presence or ab- sence of compound 5 (1:2 or 1:8 peptide/5 ratio) and incubated for 5 days at 37 8C. After this time each sample (10 mL) was dropped on the electron micros- copy grid and stained with uranyl acetate. The self-aggrega- tion in the b-sheet structure of PrP106-126 alone is evident in Figure 5 A and consisted of a dense meshwork of straight, unbranched, mature fibers. In contrast, when the peptide was co-incubated with com- pound 5, a marked reduction of the amyloid fibril formation was observed. In particular, the treatment resulted in a signifi- cant reduction in the number of mature fibers and the appear- ance of electron-dense amorphous structures instead of fibrils (Figure 5 B,C). The effect appears to be dose-dependent, as in- dicated by comparing two different peptide/compound 5 ratios (B and E, 1:2 ; C and F, 1:8).

These preliminary data highlight the ability of molecule 5 to interfere with the aggregation pathway that leads to the for- mation of PrP106-126 fibrils, which suggests a potential appli- cation as an anti-amyloidogenic compound.

Compound 5 presents some structural similarities to tetracy- cline, which, as mentioned previously, was reported to possess an inhibitory activity on a variety of amyloidogenic proteins. Nevertheless, one of the main objections to its therapeutic effi- cacy for the treatment of amyloidosis arises from the fact that the majority of patients affected by these diseases are aging, and a general improvement in their health conditions could simply spring from the antibiotic activity of these drugs. In this context, to evaluate the real therapeutic effect of new anti- amyloidogenic molecules, the identification of compounds that present, in front of a structural analogy with tetracycline, an anti-amyloidogenic but not antibacterial activity is funda- mental.

Conformational analysis, performed using molecular me- chanics (MM) and molecular dynamics (MD) simulations, shows that despite the different nature of the substituents, two of the three fused rings of our compounds present the same con- formation of rings A and B in tetracycline. In addition, a third aromatic ring is present in both structures. Calculations were performed by using the MM3*[24] force field, as implemented in the MacroModel program[25] (Maestro Suite). Conformations calculated for tetracycline and compound 5 are reported in Figure 6.

According to data collected on both Ab and PrP peptides, the lack of the fourth ring does not affect the ability of our li- gands to bind and inhibit the formation of aggregates. Howev- er, based on structure-activity relationship studies done on tet- racyclines, all four rings are essential for their antibacterial ac- tivity.[13] Therefore, a lack of antibiotic activity is expected. To assess this point, the in vitro antibacterial activity of com- pounds 1-6 was tested against an array of microorganisms consisting of three Gram-negative strains (Pseudomonas aerugi- nosa, Escherichia coli K12 wild type, and a hyperpermeable mutant strain)[26] and two Gram-positive strains (Staphylococcus aureus and Staphylococcus epidermidis). The minimum inhibito- ry concentration (MIC), defined as the lowest concentration of compound that inhibits bacterial growth after overnight incu- bation, was determined using the microbroth dilution method.[27] As a reference, tetracycline was used in the same range of concentrations. No antibacterial activity against the microorganisms tested was observed up to 0.5 mm concentra- tion of all six compounds (Table 1).

These experiments demonstrated that none of the synthetic molecules has antibacterial activity. Thus, given the com- pound 5 inhibitory activity against the formation of PrP fibrils, this molecule can be considered as a hit compound for the de- velopment of a new anti-amyloidogenic drug.

Fluorescence staining of PrP106-126 deposits As STD NMR-based epitope mapping clearly showed the mar- ginal role of the sugar moiety for the binding to amyloid oligo- mers, the hydroxyl groups may be variously functionalized to modulate our ligand hydro-/lipophobicity properties and for the conjugation to a second molecular entity. In particular, we have recently designed and synthesized compound 7,[16] which was obtained through the functionalization of the primary hy- droxyl group with a short triethylene glycol spacer allowing linkage to the 7-hydroxycoumarin fluorophore (Scheme 2 ; for the synthesis, see the Supporting Information).

Owing to the 7-hydroxycoumarin properties, molecule 7 emits blue fluorescence and has already been employed to stain Ab amyloid deposits.[16] Nevertheless, the choice of cou- marin as fluorophore was driven not only by its photophysical properties, but also by the necessity of modulating the hydro- philic properties of the conjugate, and maintaining the molec- ular weight low enough for a diffusion mechanism through the BBB. The BBB prevents the paracellular passage of the small molecules that should enter the brain by exploiting a transcellular mechanism, and represents the major obstacle to drugs targeting diseases affecting the central nervous system.[28] Only molecules with the right hydro-/lipophilic bal- ance can cross the BBB through a passive diffusion mechanism, the efficiency of which depends on their blood/brain concentra- tion gradient and their lipid solu- bility, which is hindered by a high degree of ionization and a molecular weight higher than 600 Da. Through fluorescence measurements, we have already demonstrated the capability of compound 7 of passing through an in vitro model of the BBB, most probably by exploiting a diffusion mechanism.[16] Given its ability to cross the BBB, we checked the ability of compound 7 to recognize and bind amyloid fibrils generated by peptide PrP106-126, to evalu- ate its potential use as a staining agent for prion deposits. First of all we verified, through STD NMR experiments, that 7-hydrox- ycoumarin conjugate 7 effective- ly retains the binding properties toward PrP106-126 (Figure 7). The study was performed as de- scribed for compounds 1-6.

The presence of several resonances of compound 7 in the STD NMR spectra (Figure 7) is unequivocal evidence of interac- tion with PrP106-126 oligomers. The STD NMR-based epitope mapping (Figure 8) clearly shows that both the aromatic enti- ties contribute substantially to the binding to PrP106-126 olig- omers, whereas the linker and the sugar ring play only a mar- ginal role.

The ability of compound 7 to bind and stain PrP106-126 fi- brils was then tested by fluorescence microscopy experiments. To obtain PrP106-126 fibrils, the peptide was incubated at a concentration of 0.5 m m in PBS buffer at 37 8 C for 5 days. Fibers were recovered from the sample by centrifugation at 13 000 rpm for 5 minutes and then resuspended in the pres- ence of an equimolar concentration of compound 7 for 15 mi- nutes at room temperature. Then, the pellet was washed sev- eral times to remove compound 7 in excess and analyzed by using fluorescence microscopy (Figure 9).

The aggregates showed an evident blue fluorescence emis- sion. As they are not intrinsically fluorescent, the fluorescence observed is clear evidence of the ability of compound 7 to label PrP fibrils. This was confirmed by the measurement of specific binding of thioflavine T to amyloid fibril aggregates formed by PrP106-126. These results suggested that com- pound 7 is able to recognize the b-pleated sheet secondary structure of PrP fibrils and, being also able to cross the BBB, it can be exploited as a hit compound for the design of new di- agnostic tools able to reveal the presence of amyloid aggre- gates.

Conclusion We have screened a small library of cis-glyco-fused benzopyran compounds (1-6) and have identified new ligands of PrP106- 126 oligomers. The same molecules were synthesized and characterized previously by our group as Ab ligands. They present a structural similarity to tetracycline, a natural com- pound reported to be not only a very well known antibiotic drug, but also an inhibitor of a variety of amyloidogenic pro- teins.

According to our experimental data, compounds 1-6 do not possess the characteristic antibiotic activity of tetracycline, con- sidered as an undesirable feature when evaluating the real anti-amyloidogenic activity of a potential new drug. Neverthe- less, they retain an analogous conformation of at least two of the three fused rings and, in the absence of polar substituents on the aromatic ring, also the ability to recognize and bind Ab and PrP106-126 oligomers. In addition, one of the best ligands, compound 5, was shown to be able to inhibit PrP106-126 from further aggregation. The same inhibitory activity has never been tested for Ab peptides. Moreover, the fluorescent 7-hydroxycoumarin conjugate, compound 7, has been em- ployed for the staining of PrP106-126 deposits.

Overall, the results highlight molecules 1-7 as hit com- pounds for the development of new therapeutic and diagnos- tic tools for Alzheimer's disease and mammalian prion diseas- es.

In addition, we have described an efficient experimental pro- tocol, based on NMR spectroscopy, which allows the screening of small libraries of compounds for the identification of PrP peptide ligands. To the best of our knowledge this is the first example of the application of STD NMR experiments for this purpose.

Experimental Section General All the compounds described herein were synthesized in our labo- ratory and purified by standard flash chromatography techniques. The ligand resonances were assigned by 1H, 1H,1H COSY, and 1H,13CHSQC NMR spectroscopy.[15] Peptide synthesis and purification The peptide PrP106-126 (KTNMKHMAGAAAAGAVVGGLG) was syn- thesized by solid-phase chemistry on an Applied Biosystem 433A synthesizer using 9-fluorenylmethoxycarbonyl (Fmoc)-protected l- amino acid derivatives as described previously.[29] The peptide was cleaved from the resin with phenol/thioanisole/trifluoroacetic acid, precipitated, and purified by semipreparative C4 column chroma- tography (Waters). Peptide identity was confirmed by MALDI-TOF analysis (model Reflex III, Bruker). Peptide purity was always above 95%.

NMR spectroscopy experiments All the experiments were recorded with a Bruker Avance III (600 MHz) instrument equipped with a cryoprobe. Immediately before use, lyophilized PrP106-126 was dissolved in D2O at a con- centration of 190 mm, then diluted 1:1 with 20 mm phosphate-buf- fered saline (pH 7.4) containing 100 mm NaCl (PBS) and one of the test compounds. Compounds 1-7 were dissolved in PBS, pH 7.4, and added to the peptide solution. The pH of each sample was verified with a microelectrode (Mettler Toledo) in 5 mm NMR tubes and adjusted with NaOD or DCl. All pH values were corrected for isotope effect. Basic sequences were employed for 2D TOCSY, 2D NOESY, and STD NMR experiments. For 2D TOCSY and 2D NOESY, samples were dissolved in nondeuterated PBS (H2O/D2O 9 :1) ; water suppression was achieved by excitation sculpting. A basic STD NMR sequence was used with the on-resonance frequen- cy of ^0.5 ppm and the off-resonance frequency of 40 ppm. A train of Gaussian-shaped pulses of 50 ms each was employed, with total saturation times of the protein envelope ranging from 4.0 to 0.5 s. The total saturation time was adjusted by the number of shaped pulses. Experiments were performed at 5 8C. Total sample volumes were 560 mL. The on- and off-resonance spectra were ac- quired in an interleaved mode with the same number of scans. The STD NMR spectrum was obtained by subtraction of the on-res- onance spectrum from the off-resonance spectrum. Reference ex- periments with samples containing only the free compounds tested were performed under the same experimental conditions to verify true ligand binding. The effects observed in the presence of the protein were a result of true saturation transfer, because no signals were present in the STD NMR spectra obtained in the refer- ence experiments, except for residues from HDO, thus indicating that artifacts from the subtraction of compound signals were negli- gible.

Transmission electron microscopy PrP106-126 was incubated at a concentration of 0.5 mm in PBS (pH 7.4) with or without compound 5 at two different ratios (pep- tide/compound 1:2 or 1:8). Each solution (10 mL) was dropped onto nickel formvar/carbon-coated 200 mesh electron microscopy grids (Electron Microscopy Science, Hatfield, PA, USA) and after 5 min the solution was removed. Samples were stained for 5 min with a saturated solution of uranyl acetate.[30] Electron microscopy analyses were performed with a Libra 120 transmission electron microscope (Carl Zeiss SMT, Gottingen, Germany) operating at 120 kV and equipped with a Proscan Slow Scan CCD camera (Carl Zeiss SMT).

Molecular mechanics and molecular dynamics calculations MM and MD studies were conducted with MacroModel 9.8.207 as implemented in version 9.1.207 of the Maestro suite, using the MM3* force field. The starting coordinates for dynamics calcula- tions were those obtained after energy minimization of the struc- tures, followed by conformational search. In particular, a systematic variation of the torsional degrees of freedom of the molecules per- mitted different starting structures to be constructed that were fur- ther minimized to provide the corresponding local minima. For each compound the conformer with the lowest energy was consid- ered. Simulations were performed over 5 ns at 298 K with a 0.25 fs time step and a 20 ps equilibration step; 100 structures were sam- pled and minimized for further analysis. The continuum GB/SA sol- vent model was employed and the general PRCG (Polak-Ribiere conjugate gradient) method for energy minimization was used. An extended cutoff was applied and the SHAKE procedure for bonds was not selected.

Minimal inhibitory concentration determination MIC values were determined by following standard protocols for testing susceptibility to antibiotic agents. The bacterial strains used were : Pseudomonas aeruginosa PAO1 (Gram negative), Escherichia coli K12 wild-type strain MG1655 (Gram negative), the permeable E. coli mutant strain AS19 (Gram negative), Staphylococcus aureus (Gram positive), and Staphylococcus epidermidis (Gram positive). The procedure for a broth microdilution test was followed. Briefly, 96-well microtiter plates were prepared with compound concentra- tions ranging from 500 to 0.5 mm, in triplicate. As a positive control, tetracycline hydrochloride (SIGMA) was used at the same concen- trations. The assayed strains were grown in Mueller-Hinton broth (MHB) and inoculums corresponding to an optical density at 600 nm (OD600) of 0.05 were added to each well. The microtiter plates were incubated for 18-20 h at 37 8C and the MIC of each compound determined. MIC was defined as the lowest concentra- tion inhibiting bacterial growth.

Fluorescence microscopy The peptide PrP106-126 was suspended in 100 mm phosphate buffer (pH 7.4) at a concentration of 0.5 mm, and incubated for 5 days at 37 8 C. Peptide fibers were recovered by centrifugation at 13 000 rpm for 5 min, resuspended, and incubated with com- pound 7 at room temperature in the same buffer described previ- ously. After 15 min the sample was centrifuged at 13 000 rpm for 5 min and the pellet was washed several times with phosphate buffer and applied to gelatin-coated slides. The samples were then examined by fluorescence microscopy using a DAPI filter (Zeiss).

Acknowledgements The research leading to these results received funding from Re- gione Lombardia, Fondo per la promozione di accordi istituzio- nali, Progetto no. 4779 "Network Enabled Drug Design (NEDD)" and Banca Intesa Sanpaolo. We also acknowledge Flamma (Italy) for the kind gift of Fmoc amino acids. F.C. kindly acknowl- edges Fundacao para a Ciencia e Tecnologia (FCT, Portugal) for the PhD grant (SFRH/44888/2008).

This article is part of the "Early Career Series". To view the complete series, visit : http ://chempluschem.org/earlycareer.

[1] a) G. G. Glenner, C. W. Wong, Biochem. Biophys. Res. Commun. 1984, 120, 885 - 890 ; b) C. Airoldi, E. Sironi, B. La Ferla, F. Cardona, F. Nicotra, Curr. Bioact. Compd. 2011, 7, 198 - 213.

[2] a)S.B. Prusiner, Science 1982, 216, 136-144; b)R. Zahn, A. Liu, T. Luhrs, R. Riek, C. von Schroetter, F. Lopez Garcia, M. Billeter, L. Calzolai, G. Wider, K. Wuthrich, Proc. Natl. Acad. Sci. USA 2000, 97, 145 - 150.

[3] M. G. Spillantini, M. L. Schmidt, V. M. Lee, J. Q. Trojanowski, R. Jakes, M. Goedert, Nature 1997, 388, 839 - 840.

[4] G. J. Cooper, A. C. Willis, A. Clark, R. C. Turner, R. B. Sim, F. B. Reid, Proc. Natl. Acad. Sci. USA 1987, 84, 8628 - 8632.

[5] M. Sunde, L. C. Serpell, M. Bartlam, P. E. Fraser, M. B. Pepys, C. C. Blake, J. Mol. Biol. 1997, 273, 729 - 739.

[6] M. P. Lambert, A. K. Barlow, B. A. Chromy, C. Edwards, R. Freed, M. Liosa- tos, T. E. Morgan, I. Rozovsky, B. Trommer, K. L. Viola, P. Wals, C. Zhang, C. E. Finch, G. A. Krafft, W. L. Klein, Proc Natl Acad Sci. USA 1998, 95, 6448-6453.

[7] P. Walsh, S. Sharpe, Understanding of Neurodegenerative Diseases, InTech, Croatia, 2011, pp. 89 - 214.

[8] R. Tycko, Annu. Rev. Phys. Chem. 2011, 62, 279 - 299.

[9] a) S. Chimon, M. A. Shaibat, C. R. Jones, D. C. Calero, B. Aizezi, Y. Ishii, Nat. Struct. Mol. Biol. 2007, 14, 1157 - 1164 ; b) C. Airoldi, E. Sironi, B. La Ferla, F. Cardona, F. Nicotra, Curr. Bioact. Compd. 2011, 7, 198 - 213.

[10] P. Walsh, P. Neudecker, S. Sharpe, J. Am. Chem. Soc. 2010, 132, 7684 - 7695.

[11] a) C. G. Glabe, Trends Biochem. Sci. 2004, 29, 542 - 547; b) C. M. Dobson, Nature 2003, 426, 884 - 890 ; c) A. Aguzzi, M. Heikenwalder, M. Polymeni- dou, Nat. Rev. Mol. Cell Biol. 2007, 8, 552 - 56.

[12] B. Y. Feng, B. H. Toyama, H. Wille, D. W. Colby, S. R. Collins, B. C. H. May, S. B. Prusiner, J. Weissman, B. K. Shoichet, Nat. Chem. Biol. 2008, 4, 197 - 199.

[13] For a comprehensive review, see : T. Stoilova, L. Colombo, G. Forloni, F. Tagliavini, M. Salmona, J. Med. Chem. 2013, 56, 5987 - 6006.

[14] a) F. Re, C. Airoldi, C. Zona, M. Masserini, B. La Ferla, N. Quattrocchi, F. Nicotra, Curr. Med. Chem. 2010, 17, 2990 - 3006 ; b) P. A. Novick, D. H. Lopes, K. M. Branson, A. Esteras-Chopo, I. A. Graef, G. Bitan, V. S. Pande, J. Med. Chem. 2012, 55, 3002 - 3010 ; c) P. Cavaliere, J. Torrent, S. Prigent, V. Granata, K. Pauwels, A. Pastore, H. Rezaei, A. Zagari, Biochim. Biophys. Acta 2013, 1832, 20 - 28 ; d) S. Lee, X. Zheng, J. Krishnamoorthy, M. G. Savelieff, H. Min Park, J. R. Brender, J. Hoon Kim, J. S. Derrick, A. Kochi, H. Jin Lee, C. Kim, A. Ramamoorthy, M. T. Bowers, M. Hee Lim, J. Am. Chem. Soc. 2014, 136, 299 -310.

[15] C. Airoldi, F. Cardona, E. Sironi, L. Colombo, M. Salmona, A. Silva, F. Nico- tra, B. La Ferla, Chem. Commun. 2011, 47, 10266 - 10268.

[16] C. Airoldi, F. Cardona, E. Sironi, L. Colombo, M. Salmona, I. Cambianica, F. Ornaghi, G. Sancini, F. Nicotra, B. La Ferla, Pure Appl. Chem. 2013, 85, 1813-1823.

[17] C. Airoldi, L. Colombo, C. Manzoni, E. Sironi, A. Natalello, S. M. Doglia, G. Forloni, F. Tagliavini, E. Del Favero, L. Cantu, F. Nicotra, M. Salmona, Org. Biomol. Chem. 2011, 9, 463 -472.

[18] C. Airoldi, C. Zona, E. Sironi, L. Colombo, M. Messa, D. Aurilia, M. Gre- gori, M. Masserini, M. Salmona, F. Nicotra, B. La Ferla, J. Biotechnol. 2011, 156, 317 - 324.

[19] C. Airoldi, E. Sironi, C. Dias, F. Marcelo, A. Martins, A. P. Rauter, F. Nicotra, J. Jim^nez-Barbero, Chem. Asian J. 2013, 8, 596 - 602.

[20] B. Meyer, T. Peters, Angew. Chem. 2003, 115, 890 -918; Angew. Chem. Int. Ed. 2003, 42, 864- 890.

[21] a) M. Mayer, B. Meyer, Angew. Chem. 1999, 111, 1902 -1906; Angew. Chem. Int. Ed. 1999, 38, 1784 - 1788 ; b) F. Peri, C. Airoldi, S. Colombo, S. Mari, J. Jim^nez-Barbero, M. Martegani, F. Nicotra, Eur. J. Org. Chem. 2006, 3707 - 3720 ; c) C. Airoldi, A. Palmioli, A. D'Urzo, S. Colombo, M. Vanoni, E. Martegani, F. Peri, ChemBi°Chem 2007, 8, 1376- 1379; d) A. Palmioli, E. Sacco, C. Airoldi, F. Di Nicolantonio, A. D 'Urzo, S. Shirasawa, T. Sasazuki, A. Di Domizio, L. De Gioia, E. Martegani, A. Bardelli, F. Peri, M. Vanoni, Biochem. Biophys. Res. Commun. 2009, 386, 593 - 597; e) C. Airoldi, S. Sommaruga, S. Merlo, P. Sperandeo, L. Cipolla, A. Polissi, F. Nicotra, Chem. Eur. J. 2010, 16, 1897 - 1902 ; f) R. Caraballo, H. Dong, J. P. Ribeiro, J. Jim^nez-Barbero, O. Ramstrom, Angew. Chem. 2010, 122, 599-603; Angew. Chem. Int. Ed. 2010, 49, 589-593; g) C. Airoldi, S. Sommaruga, S. Merlo, P. Sperandeo, L. Cipolla, A. Polissi, F. Nicotra, ChemBi°Chem 2011, 12, 719 - 727; h) C. Airoldi, S. Giovannardi, B. La Fer- la, J. Jim^nez-Barbero, F. Nicotra, Chem. Eur. J. 2011, 17, 13395 - 13399.

[22] J. Yan, A. D. Kline, H. Mo, M. J. Shapiro, E. R. Zartler, J. Magn. Reson. 2003, 163, 270 - 276.

[23] Y.-S. Wang, D. Liu, D. F. Wyss, Magn. Reson. Chem. 2004 , 42, 485 - 489.

[24] a) N. L. Allinger, Y. H. Yuh, J. H. Lii, J. Am. Chem. Soc. 1989, 111, 8551 - 8566 ; b) M. Mart^n-Pastor, J. F. Espinosa, J. L. Asensio, J. Jim^nez-Bar- bero, Carbohydr. Res. 1997, 298, 15 - 49.

[25] MacroModel, MacroModel 9.6, Schrçdinger, LLC, New York, 2008.

[26] M. Sekiguchi, S. Iida, Proc. Natl. Acad. Sci. USA 1967, 58, 2315 - 2320.

[27] I. Wiegand, K. Hilpert, R. E. Hancock, Nat. Protoc. 2008, 3, 163 - 175.

[28] R. Cecchelli, B. Dehouck, L. Descamps, L. Fenart, V. Bu^e-Scherrer, C. Duhem, S. Lundquist, M. Rentfel, G. Torpier, M. P. Dehouck, Adv. Drug Delivery Rev. 1999, 36, 165 - 178.

[29] a) G. Forloni, N. Angeretti, R. Chiesa, E. Monzani, M. Salmona, O. Bugiani, F. Tagliavini, Nature 1993, 362, 543 - 546 ; b) F. Tagliavini, F. Prelli, L. Verga, G. Giaccone, R. Sarma, P. Gorevic, B. Ghetti, F. Passerini, E. Ghi- baudi, G. Forloni, M. Salmona, O. Bugiani, B. Frangione, Proc. Natl. Acad. Sci. USA 1993, 90, 9678 - 9682.

[30] F. Tagliavini, G. Forloni, L. Colombo, G. Rossi, L. Girola, B. Canciani, N. Angeretti, L. Giampaolo, E. Peressini, T. Awan, L. De Gioia, E. Ragg, O. Bugiani, M. Salmona, J. Mol. Biol. 2000, 300, 1309 - 1322.

Received : January 31, 2014 Revised : March 7, 2014 Published online on June 5, 2014 Silvia Merlo,[a] Erika Sironi,[a] Laura Colombo,[b] Francisco Cardona,[a, c] Alessandra M. Martorana,[a] Mario Salmona,[b] Barbara La Ferla,[a] and Cristina Airoldi*[a] [a] Dr. S. Merlo,+ Dr. E. Sironi,+ Dr. F. Cardona, Dr. A. M. Martorana, Dr. B. La Ferla, Dr. C. Airoldi Department of Biotechnology and Biosciences University of Milano-Bicocca Piazza della Scienza 2, 20126 Milan (Italy) Fax: (+ 39) 0264483565 E-mail : [email protected] [b] Dr. L. Colombo, Dr. M. Salmona Department of Biochemistry and Molecular Pharmacology IRCCS-Istituto di Ricerche Farmacologiche Mario Negri Via La Masa 19, 20156 Milan (Italy) [c] Dr. F. Cardona Department of Chemistry, University of Aveiro Campus Universitario de Santiago, 3810-193 Aveiro (Portugal) [+] These authors contributed equally to this work.

Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cplu.201400035..

(c) 2014 Blackwell Publishing Ltd.

[ Back To TMCnet.com's Homepage ]