Molecular Design And Modification Of Two Types Of Protein Inhibitors Associated With Alzheimer’s Disease

Alzheimer’s disease（AD）is a neurodegenerative disease.The content reduction of endoneural acetylcholine and the extracellular deposition ofβ-amyloid（Aβ）are two important causes for its formation at the pathological level.Two types of proteins implicated are acetylcholinesterase and Aβpeptide.Targeting to them,various efforts have been made in the development of drugs in order to delay AD development and treat AD.Among them,rivastigmine is one of the four anti-AD drugs approved by the United States Food and Drug Administration.It is a drug with anti-acetylcholinesterase activity,that can delay and treat AD.The disadvantage of rivastigmine is also obvious,too.It will lead to serious dose-response relationship and other problems.That is,higher dosage is usually required for effective treatment clinically against acetylcholinesterase activity,the incidence of side effects will go up,however.There are two types of inhibitors in preventing Aβdeposition.One is the inhibitor for Aβaggregation induced by Cu²⁺.Such inhibitors（e.g.CQ3）have metal-chelating properties and good hydrophilicity,but are not effective in inhibiting Aβself-aggregation.Another are those for Aβself-aggregation,such as ER,TS0,etc.,which are characterized by good hydrophobicity and prone to bind the hydrophobic nucleation region of Aβ.No metal-chelation region in these inhibitors blocks the inhibition effect on the metal-induced Aβaggregation.Therefore,this thesis focused on design and modification of drugs/inhibitors,and carried out two aspects of research work（design of novel inhibitors,modification of the wild inhibitors）.（1）Design of novel inhibitors:By constructing the QSAR model,two inhibitors with higher inhibitory activity for acetylcholinesterase were screened and designed,the inhibitory activity of inhibitors at unit concentration was increased as expected.We also disclosed the interaction mechanism and observed key binding sites between inhibitors and acetylcholinesterase;（2）For modification of the wild inhibitors,three modified small molecules E1C,E2C and TQ were obtained by recombining inhibitors CQ3 and ER/CQ3 and TS0,characterized by containing both hydrophilic metal-chelating group and large hydrophobic group.These modified molecules are expected to enable to inhibit Aβ42 self-aggregation and metal-induced aggregation.as they inherit the outstanding advantages of both sides in inhibiting Aβ42 aggregation and chelating metal ions from their parental molecules.Finally,the interaction between three modified small molecules and Cu²⁺-Aβ42system,and the underlying mechanism were studied.The effects of three modified small molecules on the oxidation of Cu²⁺-Aβ42 complex were discussed,and both the advantages and disadvantages of modified small molecules were analyzed.Regarding the design of inhibitors with higher acetylcholinesterase inhibitory activity:Three-dimensional quantitative structure-activity relationship（3D-QSAR）,molecular docking and molecular dynamics simulation strategies were employed to screen and design novel inhibitors,and two candidates L-1 and L-2 with higher inhibitory activity（their inhibitory activity was increased by about 30%and 34%,respectively,relative to rivastigmine）were obtained successfully.It was found that group with electron-donating property could improve the inhibitory activity of inhibitors.Topomer Co MFA model with good predictive ability was established by 3D-QSAR model and combining with 25 compounds reported in the experiment（the non-cross validation coefficient was r²=0.902）.The model accurately verified the rationality of the predictive value for the inhibitory activity of L-1 and L-2.Further studies showed that binding sites accommodated by cetylcholinesterase for targeting were the same as those observed for rivastigmine in the experiment,but with different binding strength,i.e.,either L-1 or L-2 binds cetylcholinesterase stronger than rivastigmine.This work demonstrates a design strategy to enhance the inhibitory activity of carbamate inhibitors using electron-donating groups,which is expected to simplify the design process of complicated bioactive molecules.Modified inhibitors of Cu²⁺-Aβ:CQ3 compound is an effective chelating agent for metal ions,but for hydrophobic regions to inhibit the self-aggregation of Aβdue to its high hydrophilicity.Since erythrosine（ER）and tanshinone derivatives（TS0）have larger hydrophobic groups,they can better act on the hydrophobic region of Aβand therefore inhibit Aβself-aggregation,but Cu²⁺-induced Aβaggregation.Therefore,we recombined above three molecular structures to obtain three modified small molecules,two negatively charged E1C and E2C,and a neutral TQ,so that they can collect both fortes,possessing both metal-chelating property and hydrophobicity.The simulation results showed that the three modified small molecules had indeed stronger inhibition on Cu²⁺induced Aβ42 misfolding than CQ3.Under faintly acidic conditions,TQ is the most effective inhibitor,which not only increases the helical content of Cu²⁺-Aβ42,but also improves the oxidation ability.Under alkalescent condition,two charged molecules E1C and E2C increase both the helical content of Cu²⁺-Aβand oxidation activity,suggesting that the inhibition effect of two charged molecules depends on a variety of factors,including at least the reduced helical structure and increased oxidation activity.Modified small molecules containing neutral and larger hydrophobic aromatic groups are more effective in inhibiting Cu²⁺induced Aβ42 aggregation under both alkalescent and faintly acidic conditions.This work shows that the three modified small molecules have stronger inhibition ability to Cu²⁺-Aβ42 aggregation than their parental molecules（CQ3,ER,TS0）.At the same time,the possible mechanism of action and the effects of various influencing factors are revealed at the molecular level,which provides a reference for the improvement of inhibitors in the future.