Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma

Transthyretin (TTR) tetramer dissociation and misfolding facilitate assembl y into amyloid fibrils that putatively cause senile systemic amyloidosis an d familial amyloid polyneuropathy. We have previously discovered more than 50 small molecules that bind to and stabilize tetrameric TTR, inhibiting am yloid fibril formation in vitro. A method is presented here to evaluate the binding selectivity of these inhibitors to TTR in human plasma, a complex biological fluid composed of more than 60 proteins and numerous small molec ules. Our immunoprecipitation approach isolates TTR and bound small molecul es from a biological fluid such as plasma, and quantifies the amount of sma ll molecules bound to the protein by HPLC analysis. This approach demonstra tes that only a small subset of the inhibitors that saturate the TTR bindin g sites in vitro do so in plasma. These selective inhibitors can now be tes ted in animal models of TTR amyloid disease to probe the validity of the am yloid hypothesis. This method could be easily extended to evaluate small mo lecule binding selectivity to any protein in a given biological fluid witho ut the necessity of determining or guessing which other protein components may be competitors. This is a central issue to understanding the distributi on, metabolism, activity, and toxicity of potential drugs.