Search of ligands for the amyloidogenic protein β2-microglobulin by capillary electrophoresis and other techniques

Abstract

Beta2-microglobulin (beta2-m) is a small amyloidogenic protein normally present on the surface of most nucleated cells and 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 beta2-m through the binding to a small molecule, and consequent inhibition of protein misfolding and amyloid fibril formation. A few compounds have been described to weakly bind beta2-m, including the drug suramin. The lack of a binding site for nonpolypeptidic ligands on the beta2-m structure makes it difficult for both the identification of functional groups responsible for the binding and the search of hits to be optimized. The characterization of the binding properties of suramin for beta2-m by using three different techniques (surface plasmon resonance, affinity CE (ACE), ultrafiltration) is here described and the results obtained are compared. The common features of the chemical structures of the compounds known to bind the protein led us to select 200 sulfonated/suramin-like molecules from a wider chemical library on the basis of similarity rules, so as to possibly single out some interesting hits and to gain more information on the functional groups involved in the binding. The development of screening methods to test the compounds by using ultrafiltration and ACE is described.

Surface Nanomechanics of Biomolecules and Supramolecular Systems

“…M) [29,30], but only congo red exerts an in vitro antiamyloid activity [28], matching the nanomechanical results.…”

“…activity of the selected set of small ligands, the β2-m MCs were incubated ex situ in a 6 μM solution of congo red, suramin or nonbinder (see Ref. [29] for the ligand concentration). Then, the β2-m MCs system was exposed again to the pH switch.…”

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

“…The affinity screening of 200 sulfonated molecules previously carried out by ACE offered a wide choice of structures unable to form complexes with b 2 -m. [21,22] Therefore, to ensure that the ESI-MS method described herein is able to detect noncovalent b 2 -m complexes formed in the condensed phase and to exclude formation of nonspecific aggregates in the gas phase, we selected three known nonbinders (namely 874, 631, and 499) that nevertheless possess some chemical features of suramin, and in particular the sulfonated residues and the aromatic rings which are potentially able to form electrostatic and hydrophobic interactions with the target protein in the gas phase. None of the tested compounds were found to form noncovalent b 2 -m complexes as the deconvoluted mass spectra at the end of the incubation time were superimposable with those obtained by incubating b 2 -m alone and no peaks at the M r of predicted complexes were detected.…”

“…The binding properties of suramin for b 2 -m have been previously characterised by us, using three complementary techniques, namely ACE in the Dynamic Complexation Capillary Electrophoresis mode, SPR, and ultrafiltration. [21] Notably, only the ACE data could address the partially structured folding intermediate as a molecular target, as it is electrophoretically separated in solution and therefore independent from the native form of b 2 -m. Although a characterisation by ESI-MS of the early dynamic stages of b 2 -m fibrillogenesis, including pre-amyloid oligomer formation, has been recently reported, [5,8,26] it is beyond the scope of this work to address these processes. Affinity data herein are therefore described considering b 2 -m in solution, under non-denaturing conditions in the absence of any agent that would favour b 2 -m aggregation.…”

“…We started from the few molecules that were known to bind the native form of b 2 -m, including Congo Red (CR), [10,18] 8-anilino-1-naphthalene sulfonic acid, [19] and heparin, [20] although none of these sulfonated compounds had been demonstrated to have stabilising effects. By ACE and surface plasmon resonance (SPR) we found that suramin, a drug structurally similar to CR, had a weak affinity for b 2 -m [21] and we developed a screening method using ACE to select new binders, by using a chemical library composed of 200 sulfonated molecules. This led to the discovery of a new ligand, 573, and when assessing whether the binding affected protein function, native structure stabilisation, refolding kinetics, and in vitro self aggregation into amyloid fibrils, we established that a small molecule that speeds up refolding inhibits in vitro fibril formation.…”