The Inhibitory Effects Of Hematoxylin On The Aggregation Of Amyloid-? Protein

Amyloid protein misfolding leads to many diseases.Among them,accumulation and aggregation of amyloid ?-protein(A?)play an important role in the pathogenesis of Alzheimer's disease(AD).Hence,inhibition of A? aggregation is an effective therapeutic method for AD.In recent reseach,more extensive attention has been taken to the natural small molecule inhibitors because of their high permeability through the blood-brain barrier and low cytotoxicity.Yet,none of the existing inhibitors has been used for the clinical treatment of AD.Therefore,more effective natural molecules with high inhibitory capability on A? fibrillation are urgently required.Hematoxylin,a natural compound of caesalpinia sappan,was reported to inhibit the aggregation of A?42 and its cytotoxicity effectively.To explore thedetailedinhibitoryprocess of hematoxylin,its analogue brazilin,an inhibitor reported earlier,was selected as the control compound for comparative analysis.The inhibitory effects andmechanismsof hematoxylin on the aggregation and cytotoxicity of A?42 were extensively examined by a variety of experimental methods.The results demonstrated that hematoxylin was a powerful inhibitor of A?42 fibrillogenesis with an IC50 of 1.6 ?M,which was 30% less than that of brazilin(IC50 ~2.3 ?M).Notably,hematoxylin reduced the ?-sheet content of A?42(19 %)and made it assemble into antiparallel arrangement,which induced A?42 to form unstructured aggregates observed by TEM.Brazilin present a similar but lowerlevel inhibitoryeffect with hematoxylin.Asthe result,hematoxylin greatly reduced A?42-induced cytotoxicity.Molecular dynamics(MD)simulations revealed the detailed interactions between hematoxylin and A?42.Four binding sites of hematoxylin on A?42 hexamer were identified,including the N-terminal region,S8GY10 region,turn region and C-terminal region.Notably,more abundant hydroxyls made hematoxylin prefer to interact with the charged residues and formed more plenty of hydrogen bonds with A?42 than brazilin.This also contributed to the formation of ?-? stacking and hydrophobic interactions.That was the reason why hematoxylin preformed a lower IC50 than brazilin.Taken together,hematoxylin could not only reduce the stability of A?42 hexamer but also prevent the further binding of A?42 monomer.This is the inhibitory mechanism of hematoxylin.Finally,the inhibitory effects of four small moleculeinhibitors were examined,and two different inhibitory mechanisms were revealed by secondary structure analysis.EGCG protected the ?-helix of N-terminal residues,maintained the ?-helix structure of A?42 monomer and hindered on-pathway aggregation,while hematoxylin,brazilin and curcumin were able to perturb the distribution of ?-sheet on A?42 monomer,induce antiparallel arrangement of ?-sheet and redirect A?42 aggregation into unstructured,off-pathway aggregates.MD results indicated that because EGCG,yet hematoxylin perturbed the distribution of ?-sheet on A?42 monomer.The study revealed thathematoxylin was a potential compound for therapeutic treatment of AD.And the inhibitory mechanisms of hematoxylin and other inhibitorswould provide a theoretical basis for the further reseach and the rational designand screen of new inhibitors.