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

Abstract

Human beta2-microglobulin (beta2-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 beta2-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 beta2-m folding. These data are correlated with the effect that the same molecules exert on in vitro fibrillogenesis experiments.

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.…”

“…The set of small ligands were selected in order to cover the most relevant scenarios: congo red, a dye that probes fibril formation, speeds up the protein refolding kinetics and can abolish in vitro fibril deposition [28], suramin, a urea derivative which also binds the protein but does not interfere with its refolding and without antiamyloid activity [28], and a reference sulfonated molecule that does not bind the protein, hereafter referred as nonbinder. 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.…”

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 compound 573 proved to be the only molecule of the 200 previously screened by ACE that binds b 2 -m. [22] Despite its affinity constant, as calculated by ACE and SPR, being similar to that of suramin (K a = 10 3 -10 4 m…”

“…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. [22,23] Herein we use suramin, CR, 573, and three reference nonbinders that were identified from the screening experiments, to establish a complementary approach that combines highly accurate and specific affinity data using high-resolution mass spectrometry with in silico docking simulations. The aims are to confirm previous affinity data by a highly sensitive and rapid measurement, to achieve new insight on the chemical groups involved in the binding, to identify the interactions relevant to the antiamyloidogenic activity, to envisage new screening methods by both approaches, and to use the technique for predictive purposes.…”