Ortho-methylated 3-hydroxypyridines hinder hen egg-white lysozyme fibrillogenesis

Protein aggregation with the concomitant formation of amyloid fibrils is related to several neurodegenerative diseases, but also to non-neuropathic amyloidogenic diseases and non-neurophatic systemic amyloidosis. Lysozyme is the protein involved in the latter, and it is widely used as a model system to study the mechanisms underlying fibril formation and its inhibition. Several phenolic compounds have been reported as inhibitors of fibril formation. However, the anti-aggregating capacity of other heteroaromatic compounds has not been studied in any depth. We have screened the capacity of eleven different hydroxypyridines to affect the acid-induced fibrillization of hen lysozyme. Although most of the tested hydroxypyridines alter the fibrillation kinetics of HEWL, only 3-hydroxy-2-methylpyridine, 3-hydroxy-6-methylpyridine and 3-hydroxy-2,6-dimethylpyridine completely abolish fibril formation. Different biophysical techniques and several theoretical approaches are combined to elucidate their mechanism of action. O-methylated 3-hydroxypyridines bind non-cooperatively to two distinct but amyloidogenic regions of monomeric lysozyme. This stabilises the protein structure, as evidenced by enhanced thermal stability, and results in the inhibition of the conformational transition that precedes fibril assembly. Our results point to o-methylated 3-hydroxypyridines as a promising molecular scaffold for the future development of novel fibrillization inhibitors.

Supplementary Figure S15. Changes in the fluorescence intensity of HEWL measured at 340nm (λexc 280nm) as function of (A) 2m-3HP, (B) 6m-3HP or (C) 2,6dm-3HP concentrations at pH 2.0 and 37ºC. Experimental data is shown as black dots. Dashed red lines (---) represent the best theoretical fit to a model involving one protein binding site, thus forming a 1:1 protein-HP complex.
Continuous red lines ( ___ ) represent the best theoretical fit to a model involving two independent binding sites, thus forming a 1:2 protein-HP complex. The experimental data was fitted to both models using the Dynafit software taking into account the HEWL and the o-methylated 3HP dilution effects associated to the titration. Figure S16. Cartoon representation of HEWL (PDB code 1LSE). The entirely HEWL structure is shown as cartoons (grey), except the unstructured region between the helices A and B (H15-G26) which was colored in atom-coloured sticks. The side chains of the residues within this region capable to accept/donate hydrogen are also shown as atom-coloured sticks (D18, N19, R21 and N27). The image was generated using the molecular graphics software PyMOL.  The RMS values were obtained from the different fifteen conformations taking lowest energy conformation as reference. c The interaction between these residues and the ligand occurs through a hydrogen bond. d The RMS value render the variation between the structures ensemble in the cluster shown in white in Figure 5a (left). e The RMS value render the variation between the two clustered ensembles (green and brown ligands) shown in the Figure 5a (left). f The RMS value implies the variation between the two clustered ensembles found for this binding site. g The RMS value render the variation between the structures ensemble in the cluster shown in white in Figure 5c (left). h The RMS value render the variation between the structures ensemble in the cluster shown in green in Figure 5c (left).

MALDI-TOF/TOF mass spectrometry
0.5µl aliquots of solutions containing HEWL (0.2mM) previously incubated during 0 or 10d at pH 2.0 and 60ºC either alone or in the presence of 20mM 2m-3HP, 6m-3HP or 2,6dm-3HP were spotted onto a steel target plate (MTP 384), air-dried, and subjected to mass determination. Mass spectra were analyzed on a Bruker Autoflex III MALDI-TOF spectrometer equipped with a 200-Hz smart-beam pulsed N2 laser (λ 337 nm). The IS1 and IS2 voltages were 19 kV and 16.65 kV respectively, and the lens voltage was 8.2 kV. Measurements were performed using a positive reflector mode with matrix suppression below 400 Da. External calibration was performed using a standard peptide mixture. Mass spectra of digested samples were matched against Swiss-Prot databases using the Mascot search engine (Matrix Sciences).

Determination of the ionization equilibrium constants
The ionization equilibrium constants for 2m-3HP, 6m-3HP, 2,6dm-3HP, 2Cl-3HP and 5Cl-3HP were determined acquiring the corresponding absorption spectra at different pH using a Shimadzu UV-2401 PC double-beam spectrophotometer thermostated at 37ºC. Quartz cells of 1-cm path length were used to obtain the electronic spectra. Spectroscopic data were acquired over the range from 500 to 220nm. The buffer solution background spectrum was used as spectral reference. UV-Vis spectra were obtained in various 10mM buffers having a constant ionic strength of 0.5M that was adjusted by addition of KCl. The reagents used to prepare the buffer solutions were HCl (pH 1), sodium chloroacetate (pHs 2, 2.5, 3 and 3.5), sodium acetate (pHs 4, 4.5 and 5), succinic acid (pHs 6 and 6.5), potassium dihydrogen phosphate (pHs 7, 7.5 and 8), boric acid (pHs 8.5, 9 and 9.5), potassium bicarbonate (pHs 10 and 11) and NaOH (pHs 12 and 13). Each buffer was used to prepare a solution containing 0.2mM of each different 3HP. The individual UV-Vis spectra obtained at each pH were used as inputs for the factor analysis software SEPCFIT/32 TM . This allowed to obtain the characteristic UV-Vis spectrum for each ionic form and its macroscopic ionization constants.

Ab-initio calculation of electronic dipolar moment of different HPs
Standard density functional theory (DFT) calculations were carried out with the Gaussian09 software 1 . The structures corresponding to the ionic forms with a higher population at pH 2.0 were optimized for each studied HP by using the M06-2X functional in combination with the 6-311++G(d,p) basis set. Vibrational analyses were performed to verify all the optimized structures as energy minima by the absence of imaginary frequencies. All the calculations were carried out in aqueous solution modeled by the SMD solvent model 2 . The dipole moments of all HPs were calculated from the wave functions of the optimized structures in aqueous solution. In addition, wave functions were used to compute Merz-Kollman charges, Mulliken charges and the natural bond orbital charges.

Statistical analysis
The correlation between the dipolar moments computed for the main ionic forms of each HP at pH 2.0, and the fibril growth rate constant (kf) or the fibril lag-phase, were analyzed using linear regression models and Pearson correlation coefficients. A two-tailed p value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA).