Kinetic Assay for High-Throughput Screening of In Vitro Transthyretin Amyloid Fibrillogenesis Inhibitors

Abstract

Stabilization of tetrameric transthyretin (TTR) by binding of small ligands is a current strategy aimed at inhibiting amyloid fibrillogenesis in transthyretin-associated pathologies, such as senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). A kinetic assay is developed for rapid evaluation of compounds as potential in vitro inhibitors in a high-throughput screening format. It is based on monitoring the time-dependent increase of absorbance due to turbidity occurring by acid-induced protein aggregation. The method uses the highly amyloidogenic Y78F mutant of human transthyretin (heterogously expressed in Escherichia coli cells). Initial rates of protein aggregation at different inhibitor concentrations follow a monoexponential dose-response curve from which inhibition parameters are calculated. For the assay development, thyroid hormones and nonsteroidal antiinflamatory drugs were chosen among other reference compounds. Some of them are already known to be in vitro inhibitors of TTR amyloidogenesis. Analysis time is optimized to last 1.5 h, and the method is implemented in microtiter plates for screening of libraries of potential fibrillogenesis inhibitors.

Transthyretin Aggregate-Specific Antibodies Recognize Cryptic Epitopes on Patient-Derived Amyloid Fibrils

“…[18][19][20] Human amyloid b (1-40) (Ab) was synthesized and purified by Dr. James I Elliott at Yale University (New Haven, CT). A 37-mer human islet amyloid polypeptide (IAPP) was purchased from Quality Controlled Biochemicals (Hopkinton, MA).…”

“…18 Each reaction was judged complete when thioflavin T (ThT) fluorescence, a measure of amyloid fibril structure, 24 had reached a maximum plateau signal. The fibril product was isolated at room temperature by pelleting the reaction mixture, *16,000 · g for 20 min, and re-suspending the aggregates with the same volume of reaction buffer.…”

Retrospective Mapping of SAR Data for TTR Protein in Chemico‐Biological Space Using Ligand Efficiency Indices as a Guide to Drug Discovery Strategies

“…Another important point is that, these 12 compounds have the same binding pattern than iododiflunisal and all of them have the iodine atom, a possible key point to obtain selective and potent amyloid inhibitors for TTR. These molecules will be tested in fibrillogenesis inhibition studies [22] as possible new amyloidogenesis inhibitors for the TTR protein.…”

Strategies for Inhibiting Protein Aggregation: Therapeutic Approaches to Protein-Aggregation Diseases

“…Among synthetic compounds, fi ve families of compounds are prominent in the long list: bisaryloxime ethers, biphenyls, 1-aryl-4,6-biscarboxydibenzofurans, 2-phenylbenzoxazoles and biphenylamines (Oza et al , 2002Hornberg et al 2000 ;Petrassi et al 2000;Klabunde et al 2000 ;Razavi et al 2003;Adamski-Werner et al 2004 ;Purkey et al 2004;Petrassi et al 2005 ;Johnson et al 2005Johnson et al , 2008a. Many of the non-steroidal anti-infl ammatory drugs (NSAIDs), including difl unisal, and fl ufenamic and salicylic acids, have been the basis of numerous halogenated variants (Almeida et al 2004 ;Baures et al 1998Baures et al , 1999Lueprasitsakul et al 1990;Maia et al 2005 ;Adamski-Werner et al 2004 ;Miller et al 2004;Dolado et al 2005 ;Gales et al 2005 ;Mairal et al 2009). Until recently, the most potent and selective agents were the biphenyls, 2-phenylbenzoxazoles and dibenzofurans.…”