P4-169: Identification of endogenous β-amyloid anti-oligomerization agents via in silico screening of a library of known human cns compounds against the e-v-h-h-q-k domain of β-amyloid

Since many peptide sequences, including Aβ, are susceptible to amyloid-like aggregation, there is evolutionary pressure to inhibit deleterious protein misfolding; moreover, the immunoinflammatory cascade triggered by such misfolding is likewise subject to homeostatic regulation. Accordingly, it is reasonable to postulate the existence of endogenous molecules within the human brain that could modulate or even interrupt the neurotoxic cascade of AD. Such compounds would constitute platforms for therapeutic design. We sought to identify an Aβ anti-oligomerization agent endogenous to the human central nervous system; to find this compound, we created a comprehensive library of 1,110 molecules (molecular weight < 600 Da) naturally occurring within the human brain and employed an in silico screening assay. Using computer-aided screening with a molecular mechanics force field, we docked these 1,110 molecules against Aβ, identifying multiple “hits” capable of electrostatically favourable docking to the E-V-H-H-Q-K domain within Aβ. Additional molecular dynamics simulations were done to refine the docking pose and orientation of these compounds as they inhibited misfolding. Finally, multiple in vitro assays were completed, verifying that the in silico hits had correctly predicted the ability of these endogenous molecules to block oligomerization. Multiple zwitterionic compounds in which the anionic terminus interacted with the cationic “H” residues of Aβ and the cationic terminus interacted with the anionic “E” residue of Aβ were identified. Although the standard alpha amino acids showed no capacity to inhibit Aβ aggregation, a number of atypical amino acids including β-alanine, 2-aminoethanesulfonic acid, and L-phosphoserine demonstrated varying capacities to inhibit the oligomerization and aggregation of Aβ. These predictions were verified using in vitro assays, including the kinetic Thioflavin T [ThT] aggregation assay, to demonstrate the capacity of these compounds to inhibit misfolding. Searching for an “endogenous anti-AD compound” represents an unexplored concept. Our in silico and in vitro studies suggest that compounds endogenous to the human brain, including several zwitterionic small molecules, can inhibit pathological protein misfolding of Aβ. The value of a novel in silico screening assay to identify bioactive endogenous agents within brain has also been demonstrated.