Although radiotracers have been developed for imaging the cross-β-sheet aggregates appearing in neurodegenerative disease, the mechanisms that mediate their high-affinity binding (i.e., low nM) and selectivity to the surfaces presented by aggregates composed of α-synuclein (SNCA), tau, or β-amyloid are not fully understood. To clarify binding mechanism, planar heterocycles were tested in silico against 3D aggregate models using Rosetta-based docking methods. One identified binding site for a low-nanomolar affinity ligand, CC05-61, was found to engage in π-cation interaction with aggregate Lys residues. This interaction mechanism was then probed in radioligand binding assays performed on peptide models created from the PHF6 motif of tau protein, on synthetic protein aggregates prepared from recombinant monomers harboring Lys to Ala mutants generated by site directed mutagenesis, and in competition format using fluoro-substituted structure analogs designed to disrupt π-cation interactions. The results were consistent with specific Lys residues on synthetic tau and SNCA, but not on brain-derived β-amyloid aggregates, mediating high-affinity binding activity. These data support π-cation binding as one aggregate surface-exposed mode of interaction available to planar heterocycles for generating high affinity, high binding-site density, and selective binding to aggregate targets associated with neurodegenerative diseases.

Investigation of small molecule binding to cross-β-sheet surfaces

ACS Spring Meeting 2021

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