Raffaella Colombo

CE can identify small molecules that selectively target soluble oligomers of amyloid β protein and display antifibrillogenic activity

Soluble and toxic oligomers of amyloid β (Aβ) protein have been identified as the true neurotoxic species involved in Alzheimers disease and considering them as targets to inhibit Aβ aggregation might have a therapeutic value. We previously set up a CE method that enables the separation and quantification of transient oligomers of Aβ protein‐containing 42 amino acids (Aβ1–42) along the pathway leading to fibrils and we now demonstrate how this method can be successfully applied to examine the in vitro inhibitory effects of small molecules on Aβ oligomerization. To this end, we investigated mitoxantrone and pixantrone, two well‐known anticancer drugs, as well as suramin and a suramin‐like compound. By using CE, it is here shown how mitoxantrone and pixantrone either reduce or block Aβ1–42 oligomerization, while Thioflavin T spectrofluorimetric assay and transmission electron microscopy demonstrate how these two compounds also display antifibrillogenic activity. Interestingly, in vitro cell viability experiments indicated that pixantrone significantly reduces Aβ1–42 neurotoxicity.

Multifunctional cholinesterase and amyloid Beta fibrillization modulators. Synthesis and biological investigation.

In order to identify novel Alzheimers modifying pharmacological tools, we developed bis-tacrines bearing a peptide moiety for specific interference with surface sites of human acetylcholinesterase (hAChE) binding amyloid-beta (Aβ). Accordingly, compounds 2a-c proved to be inhibitors of hAChE catalytic and noncatalytic functions, binding the catalytic and peripheral sites, interfering with Aβ aggregation and with the Aβ self-oligomerization process (2a). Compounds 2a-c in complex with TcAChE span the gorge with the bis-tacrine system, and the peptide moieties bulge outside the gorge in proximity of the peripheral site. These moieties are likely responsible for the observed reduction of hAChE-induced Aβ aggregation since they physically hamper Aβ binding to the enzyme surface. Moreover, 2a was able to significantly interfere with Aβ self-oligomerization, while 2b,c showed improved inhibition of hAChE-induced Aβ aggregation.

Disease modifying anti-Alzheimer’s drugs: inhibitors of human cholinesterases interfering with β-amyloid aggregation

We recently described multifunctional tools (2a–c) as potent inhibitors of human Cholinesterases (ChEs) also able to modulate events correlated with Aβ aggregation. We herein propose a thorough biological and computational analysis aiming at understanding their mechanism of action at the molecular level.

In vitro amyloid Aβ1-42 peptide aggregation monitoring by asymmetrical flow field-flow fractionation with multi-angle light scattering detection

Self-assembly of the 42-amino-acid-long amyloid peptide Aβ1-42 into insoluble fibrillar deposits in the brain is a crucial event in the pathogenesis of Alzheimers disease. The fibril deposition occurs through an aggregation process during which transient and metastable oligomeric intermediates are intrinsically difficult to be accurately monitored and characterised. In this work, the time-dependent Aβ1-42 aggregation pattern is studied by asymmetrical flow field-flow fractionation with on-line multi-angle light scattering detection. This technique allows separating and obtaining information on the molar mass (Mr) and size distribution of both the early-forming soluble aggregates and the late prefibrillar and fibrillar species, the latter having very high Mr. Preliminary results demonstrate that unique information on the dynamic aggregation process can be obtained, namely on the Mr and size of the forming aggregates as well as on their formation kinetics.

Sulfonated molecules that bind a partially structured species of β2‐microglobulin also influence refolding and fibrillogenesis

Human β2‐microglobulin (β2‐m) is a small amyloidogenic protein responsible for dialysis‐related amyloidosis, which represents a severe complication of long‐term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of β2‐m through the binding to a small molecule, to possibly inhibit protein misfolding and amyloid fibril formation. The search of a strong ligand of this protein is extremely challenging: by using CE in affinity and refolding experiments we study the effect that previously selected sulfonated molecules have on the equilibrium between the native form and an ensemble of conformers populating the slow phase of β2‐m folding. These data are correlated with the effect that the same molecules exert on in vitro fibrillogenesis experiments.

Screening of fibrillogenesis inhibitors of β2-microglobulin: Integrated strategies by mass spectrometry capillary electrophoresis and in silico simulations

The challenging search of ligands for the amyloidogenic protein β(2)-microglobulin led us to set up an integrated strategy that combines analytical techniques and molecular modelling. Using a chemical library composed of 90 sulphonated molecules and a novel MS screening approach, we initially single out a few new binders. To check for anti-amyloid activity, the best hit obtained was thoroughly studied by docking analysis, affinity and refolding experiments by capillary electrophoresis and in vitro fibrillogenesis Thioflavin T test. Correlative analysis of the overall results obtained from the MS screening led to develop an equation able to identify the key factors of the affinity for β(2)-microglobulin and to predict the affinity for novel derivatives. The proposed equation was then used for a virtual screening of a large compound database. Studies on the new hit thus retrieved confirm the predictive potential of both the equation on affinity and of docking analysis on anti-amyloid activity.

Separation and characterisation of beta2-microglobulin folding conformers by ion-exchange liquid chromatography and ion-exchange liquid chromatography-mass spectrometry.

In this work we present for the first time the use of ion-exchange liquid chromatography to separate the native form and a partially structured intermediate of the folding of the amyloidogenic protein beta2-microglobulin. Using a strong anion-exchange column that accounts for the differences in charge exposure of the two conformers, a LC-UV method is initially optimised in terms of mobile phase pH, composition and temperature. The preferred mobile phase conditions that afford useful information were found to be 35 mM ammonium formate, pH 7.4 at 25°C. The dynamic equilibrium of the two species is demonstrated upon increasing the concentration of acetonitrile in the protein sample. Then, the chromatographic method is transferred to MS detection and the respective charge state distributions of the separated conformers are identified. The LC-MS results demonstrate that one of the conformers is partially unfolded, compared with the native and more compact species. The correspondence with previous results obtained in free solution by capillary electrophoresis suggest that strong ion exchange LC-MS does not alter beta2-microglobulin conformation and maintains the dynamic equilibrium already observed between the native protein and its folding intermediate.

A Combined High-Resolution Mass Spectrometric and in silico Approach for the Characterisation of Small Ligands of β2-Microglobulin

β2‐Microglobulin (β2‐m) is a protein responsible for a severe complication of long‐term hemodialysis, known as dialysis‐related amyloidosis, in which initial β2‐m misfolding leads to amyloid fibril deposition, mainly in the skeletal tissue. Whereas much attention is paid to understanding the complex mechanism of amyloid formation, the evaluation of small molecules that may bind β2‐m and possibly inhibit the aggregation process is still largely unexplored mainly because the protein lacks a specific active site. Based on our previous findings, we selected a pilot set of sulfonated molecules that are known to either bind or not to the protein, including binders that are anti‐amyloidogenic. We show how a complementary approach, using high‐resolution mass spectrometry and in silico studies, can offer rapid and precise information on affinity, as well as insight into the structural requisites that favour or disfavour the inhibitory activity. Overall, this approach can be used for predictive purposes and for a rapid screening of fibrillogenesis inhibitors.

Advances on Size Exclusion Chromatography and Applications on the Analysis of Protein Biopharmaceuticals and Protein Aggregates: A Mini Review

Biopharmaceutical drugs are large, complex protein molecules derived from living cells. The characterization of the protein-based products is a complicated and challenging task necessary to guarantee their efficacy and safety. A major concern in manufacturing protein biopharmaceuticals is their propensity to form aggregates. The separation of proteins by size-exclusion chromatography (SEC) is a well-established method for the characterization of macromolecules and their aggregates. The demand for increased resolution and faster analysis in SEC has led to the development of small, more rigid fully porous particles and superficially porous particles with different pore sizes. The coupling of SEC with three or four detectors has enhanced protein characterization by generating accurate molecular weights (absolute determination) and by giving insight into the degradation pathways and products associated with aggregation. The intent of this review is to report the continuous developments of SEC stationary phases and their applications on the characterization of intact proteins, their variants or aggregates and protein complexes. Thus, the most recent applications of SEC with the focus on proteins and protein aggregates are reported and discussed.

Advanced glycation end products of beta2-microglobulin in uremic patients as determined by high resolution mass spectrometry.

By using a high resolution top-down and bottom-up approach we identified and characterized the AGEs of beta2-microglobulin (β2-m) formed by incubating the protein in the presence of glucose and of the main reactive carbonyl species. Glucose induced glycation on the N-terminal residue, while glyoxal (GO) and methylglyoxal (MGO) covalently reacted with Arg3. Carboxymethyl (CM-R) and imidazolinone (R-GO) derivatives were identified in the case of GO and carboxyethyl arginine (CE-R) and methyl-imidazolinone (R-MGO) for MGO. Interestingly, α,β-unsaturated aldehydes [4-hydroxy-2-nonenal (HNE); 4-oxo-2-nonenal (ONE); acrolein (ACR)] did not induce any covalent modifications up to 100μM. The different reactivity of β2-m towards the different RCS was then rationalized by molecular modeling studies. The MS method was then applied to fully characterize the AGEs of β2-m isolated from the urine of uremic subjects. CM-R, CE-R and R-MGO were easily identified on Arg3 and their relative abundance in respect to the native protein determined by a semi-quantitative approach. Overall, the AGEs content of urinary β2-m ranged from 0.2 to 1% in uremic subjects. The results here reported offer novel insights and technical achievements for a potential biological role of AGEs-β2-m in pathological conditions.