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

Abstract

Transthyretin (TTR) tetramer dissociation and misfolding facilitate assembly into amyloid fibrils that putatively cause senile systemic amyloidosis and familial amyloid polyneuropathy. We have previously discovered more than 50 small molecules that bind to and stabilize tetrameric TTR, inhibiting amyloid 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 molecules. Our immunoprecipitation approach isolates TTR and bound small molecules from a biological fluid such as plasma, and quantifies the amount of small molecules bound to the protein by HPLC analysis. This approach demonstrates that only a small subset of the inhibitors that saturate the TTR binding sites in vitro do so in plasma. These selective inhibitors can now be tested in animal models of TTR amyloid disease to probe the validity of the amyloid hypothesis. This method could be easily extended to evaluate small molecule binding selectivity to any protein in a given biological fluid without the necessity of determining or guessing which other protein components may be competitors. This is a central issue to understanding the distribution, metabolism, activity, and toxicity of potential drugs.

Figure 1

Rabbit polyclonal anti-TTR antibodies selectively bind TTR in human blood plasma. Lane 1, molecular mass standards; lane 2, isolated human blood plasma diluted 10-fold; lane 3, recombinant human TTR. The higher molecular mass band in lane 3 is due to an SDS-resistant dimer present when the recombinant protein is run on a gel.

Figure 2

Summary of the procedure to evaluate inhibitor binding selectivity to TTR versus other plasma proteins. Human blood plasma is incubated with a small molecule inhibitor (black bars) for 24 h. The TTR tetramer (squares) and bound small molecules are precipitated with antibody-conjugated Sepharose (circle). The Sepharose is washed to remove unbound proteins (oval and diamond) and small molecules (triangle). TTR with (shown here) or without ligands bound is eluted at high pH. The supernatant is loaded onto an HPLC column for analysis and quantification.

Figure 3

HPLC chromatogram of immunoprecipitated plasma TTR in the absence of small molecule inhibitors (A) and in the presence of compound 9, a small molecule inhibitor of TTR fibril formation exhibiting selective TTR binding in plasma (B). TTR appears as two peaks, the first peak being monomer disulfide linked to cysteine and the second peak being unconjugated monomer.

Figure 4

Modest structural changes of the small molecule dramatically alter TTR plasma binding selectivity of chlorinated biarylamines possessing equal in vitro amyloid inhibition efficacy.