Associating a negatively charged GdDOTA-derivative to the Pittsburgh compound B for targeting A beta amyloid aggregates

摘要

We have conjugated the tetraazacyclododecane-tetraacetate (DOTA) chelator to Pittsburgh compound B (PiB) forming negatively charged lanthanide complexes, Ln(L-4), with targeting capabilities towards aggregated amyloid peptides. The amphiphilic Gd(L-4) chelate undergoes micellar aggregation in aqueous solution, with a critical micellar concentration of 0.68 mM, lower than those for the neutral complexes of similar structure. A variable temperature O-17 NMR and NMRD study allowed the assessment of the water exchange rate, k (ex) (298) = 9.7 x 10(6) s(-1), about the double of GdDOTA, and for the description of the rotational dynamics for both the monomeric and the micellar forms of Gd(L-4). With respect to the analogous neutral complexes, the negative charge induces a significant rigidity of the micelles formed, which is reflected by slower and more restricted local motion of the Gd3+ centers as evidenced by higher relaxivities at 20-60 MHz. Surface Plasmon Resonance results indicate that the charge does not affect significantly the binding strength to A beta(1-40) [K (d) = 194 +/- A 11 mu M for La(L-4)], but it does enhance the affinity constant to human serum albumin [K (a) = 6530 +/- A 68 M-1 for Gd(L-4)], as compared to neutral counterparts. Protein-based NMR points to interaction of Gd(L-4) with A beta(1-40) in the monomer state as well, in contrast to neutral complexes interacting only with the aggregated form. Circular dichroism spectroscopy monitored time- and temperature-dependent changes of the A beta(1-40) secondary structure, indicating that Gd(L-4) stabilizes the random coil relative to the alpha-helix and beta-sheet. TEM images confirm that the Gd(L-4) complex reduces the formation of aggregated fibrils.