Study On Anti-alzheimer's Disease Effects And Molecular Mechanism Of A Novel Compound SCR-1693

Alzheimer's disease(AD),an age-related,progressive neurodegenerative disorder,is the most common form of dementia.The major pathological hallmarks of AD brain are senile plaques,consisting predominantly of extracellular amyloid-? peptides,and neurofibrillary tangle(NFTs),consisting of polymerized hyper-phosphorylated tau protein,accompany by neuronal loss,brain hypometabolism,neuroinflammation,mitochondria dysfunction.The clinical symptoms of AD patients are characterized by progressive loss memory loss and other cognitive dysfunction,and quality-of-life impairment.So far,only four cholinesterase inhibitors(ChEIs),including tacrine,donepezil,rivastigmine and galantamine,and an N-methyl-D-aspartate(NMDA)receptor antagonist memantine have been licensed for the treatment of cognitive symptoms of AD.Unfortunately,no drugs can cure or modify the process of AD.In the present study,we investigated a new synthesized 1,4-dihydro-naphthyridine derivative SCR-1693,ethyl 5-amino-4-(2-chlorophenyl)-2,7,7-trimethyl-1,4,6,7,8,9-hexahydrobenzo[b][1,8]naphthyridine-3-carboxylate,which possesses a merged nuclear structure from tetrahydroaminoacridine(THA)and dihydropyridine(DHP)and targets both acetylcholinesterase(AChE)and calcium channels.Enzyme activity assays in vitro showed that SCR-1693 acted as a selective,reversible and non-competitive inhibitor of AChE,with an IC50 value of 0.68?M and a Ki value of 0.97 ?M,and had no imbibition on BuChE.In Wistar rats,single oral administration of SCR-1693 dose-dependently caused the inhibition of brain homogenate AChE activity ex vivo,rather than plasma AChE activity.In SH-SY5Y cells,SCR-1693 dose-dependently inhibited KCl-evoked intracellular Ca2+ increase with an IC50 value of 28.6 ?M.In rat DRG neurons,SCR-1693 at the concentrations of 100 nM and 1000 nM inhibited L-type calcium current by 38.14%and 61.76%,respectively,which showed more stronger activity than nifedipine at the same molar concentration.The blockage effects of SCR-1693 on CHO-hCav1.2(L-type),CHO-hCav2.1(P/Q-type),CHO-hCav2.2(N-type)and HEK293-hCav3.2(T-type)calcium channels indicated that SCR-1693 was a non-selective calcium channel blocker,with the inhibition of 29.7%,12.5%,18.6%and 20.8%respectively by SCR-1693 at 10 ?M.Thus,SCR-1693 acted as a selective,reversible and non-competitive inhibitor of AChE,and a non-selective voltage-gated calcium channel blocker.In A?25-35-induced SH-SY5Y cell death model,SCR-1693,at the dose of 0.02 to 2.5 ?M,reduced A?25-35-increased PI/Hoechst 33342 dying rate in SH-SY5Y cells dose-dependently,while donepezil and nilvadipine only decreased this rate at 2.5 ?M.SCR-1693 at the concentrations level of 0.1 and 0.5 ?M was more effective than not only single donepezil or nilvadipine but also their combination.These results suggested that SCR-1693 possessed a higher efficiency in decreasing A?25-35-induced SH-SY5Y cell death than donepezil or nilvadipine.In intracerebroventricular injection of the oligomer A?25-35 peptide mice(Familiar AD model),oral administration of SCR-1693 at the dosing of 0.3 and 1 mg/kg was more effective than donepezil and memantine in preventing A?25-35-induced long-term(Morris water maze)and short-term memory(Y-maze)impairment,maintaining the basal transmission of Schaffer collateral-CAl synapses,and sustaining LTP in mouse hippocampus.SCR-1693 also attenuated the loss of hippocampal pyramidal neurons and regulated A?25-35-induced signal cascade in hippocampal neurons.To investigate the underlying mechanism of SCR-1693 in regulation of tau phosphorylation and insulin resistance,we initially constructed a stable cell line,named as Neura-2a-tau,which expressed high level of phosphorylated tau and had profiles of insulin resistance.In Neura-2a-tau cells,SCR-1693 concentration-dependently decreased tau phosphorylation levels at the sites of Ser199/202,Ser202/T205,T212/Ser214,Thr231,Ser262 and Ser356,rather than Ser422,and increased tau dephosphorylation level.However,AChE inhibitor donepezil,calcium blockers(nilvadipine,nimodipine or nifedipine)or combination of AChE inhibitor and calcium blocker could not influence tau phosphorylation.These suggested that regulation of tau phosphorylation by SCR-1693 was a new mechanism,independent on inhibiting AChE or blocking VGCCs.Further study showed that SCR-1693 increased PKA and Akt activation,and increased GSK-3? phosphorylation at Ser9,deactivating of GSK-3?.However,neither of PKA inhibitor H-89 or PI3K inhibitor LY294002 could completely block SCR-1693 inhibitory effect on tau phosphorylation in Neura-2a-tau cells.These suggested that SCR-1693 regulation of tau phosphorylation was not singly dependent on PKA or Akt-GSK-3? signaling.Furthermore,we found that SCR-1693 also decreased phosphorylation of PP1 and PP2AC atThr320 and Tyr307,respectively,resulting PP1 and PP2AC activation.Treatment with calyculin A,an inhibitor both of PP1 and PP2A activity,completely inhibited SCR-1693 inhibitory effect on tau phosphorylation in Neura-2a-tau cells.From these evidence,it was deduced that SCR-1693 mainly regulated tau phosphorylation through PP1 and PP2A in Neura-2a-tau cells.Enhancement of IRS-1-PI3K-Akt signaling by SCR-1693 suggested increased insulin sensitivity of Neura-2a-tau cells,which could be restrained by PP1 and PP2A inhibitor calyculin A.In intracerebroventricular injection of STZ-induced rat model(sporadic AD model),orally administration of SCR-1693 exerted anti-amnesic effects on STZ-impaired learning and memory of STZ-injected rats.It decreased the prolongation of escape latency and increased the time of swim in the target quadrant of the pool in Morris water maze test.Consistently,tau hyperphosphorylation at sites of Ser199/202,Thr231 and Ser396,insulin signaling(IRS-1/Akt/GSK-3? signaling)impairment,astrogliosis and postsynaptic protein loss was found in hippocampus homogenous of i.c.v.-STZ injected rats.After administration of SCR-1693,these AD-like pathologies were restored.Furthermore,treatment with SCR-1693 significantly in vivo enhanced the activity of PP1 and PP2 A of hippocampus of i.c.v.-STZ rats,which was consistent with in vitro effect of SCR-1693 on Neuro-2a-rau cells.In conclusion,SCR-1693 targets on AChE and VGCCs,regulates A? and tau-associated pathology,and improves learning and memory function in animal FAD and SAD model.Thus,SCR-1693 is thought as a promising therapeutic agent for AD.