The Screening Of Compounds Against β-Amyloid (Aβ)Aggregation In Vitro And The Evaluation Of The Active Compounds

Alzheimer’s disease (AD), is an age-related neurodegenerative diseasecharacterized by the progressive loss of memory, decline in language skills, and othercognitive impairments. Pathologically, AD remains diagnosed on the basis of theformation of senile plaques (SPs), neurofbrillary tangles (NFTs) and neuro loss. Themechanism of AD remains elusive and there are no drugs to cure. By now, the drugsapproved by Food and Drug Administration (FDA) for treatment of AD includingcholinesterase inhibitors (ChEIs) and N-methyld-D-aspartate receptor (NMDAr)antagonist can only improves symptoms other than modifying the disease.Numerous hypotheses have been suggested for the etiology/pathogenesis of ADincluding genetic defects, theory of infection, inflammation theory, theory ofcholinergic, poisoning of bulk aluminum, free radical mediated processes, calciumoverload theory, Tau protein theory, theory of metabolic disorders and β-amyloid (Aβ)cascade hypothesis or some combination of the above. Amoung these hypothesises Aβcascade hypothesis is been most concerned. It’s reported in Lancet that66%of thedrug candidates against AD was targeted to Aβ in2010, furthermore to resist the Aβaggregation is the most important consern in the studyA lot of factors such asconcentrtion of Aβ, pH of the solution and metals can influence Aβ aggregation andmuch interest was payed to metal-induced Aβ aggregation. The metal chelatorclioquinol and PBT2have already showed fatastic results in animals test and theclinical trials. So our study was done to screening the inhibitors on Aβ aggregationand znic-induced Aβ aggregation from a series of metal chelator numberd HYF andsome natural product, then finally evaluate the active componds and study wheatherthe compounds can protect Aβ-induced cytotoxcity and wheather the compounds canimprove the behavior and the synaptic plasticity of dfferent types of AD mice.Section Ⅰ The establishment of screening system for inhibitors of AβaggregationTurbidity assay and Thioflavin T are now widly used in the detection of Aβaggregation. So in this part we plan to establish a fast way to detect the aggreagation ofAβ with high speed throug optimizing the two methods.1. Turbidity assayTurbidity assay can be used to detect the aggregation of Aβ by OD405. Resultsshowed that the OD405was between0.046to0.058, and there was little differencebeween different concentrtions of Aβ25-35and Aβ1-42. Moreover all the samples hadslight change against time during48h incubation. So turbidity assay could not beused in drug screening.2. Th-T binding testThioflavin T can bind to amyloid β-sheets, and upon the binding there is acharacteristic shift in the absorbance band. Under the excitation of450nm we candetect the intensity of fluencence which can represent the quantity of Aβ aggregationat485nm emission. Fluorescence detection can be performed on micro readers andfluorospectrophotometer. So in this study we first used micro reader to detect theaggregation of Aβ for its high flex. The results found that Aβ gathered over time, andafter7h incubation there was a big difference between the replicates and the repeatinspection with no time delay were found to have big difference. The reason for thismay be caused by the instrument, for the micro reader require the sample system to behomogeneous, however Aβ1-42gathered over time and the plaques became bigger,which can broke the system homogeneous. So results from micro reader could notrepresent the aggregation ofAβ correctly.Furthermore on the basis of upon binding to Th-T Aβ1-42can be seen under themicro scop, so in this study we used high-speed cell analyzer (Acumen) which has thefunction of fluorescence quantitative to do the fluorescence detection.The results showed that the fluorescence intensity of Aβ1-42(2.5-40μM) canincrease dependently with the concentrations in the Th-T binding test. Moreover whenthe concentration was less than10μM, the fluorescence intensity and concentrationhad good linear relationship (R2=0.99997), however when the maximumconcentrition was40μM the linear fitting became worse, so this indicate that whenthe concentration was lower than10μM, the the fluorescence intensity can reflectdirectly the aggregation of Aβ1-42. And10μM Aβ1-42was chosen in the followingstudy.In the time dependent increasing tests of Aβ1-42aggregation we found the numberof Aβ1-42aggregation and also the size of the aggregated Aβ1-42increased with timetThe fluorescence intensity results was the same with other reference we found thefluorescence intensity increased with time, and achieved the platform after12hincubation, after24h it tended to be stable, so this results indicate that in the sameincubation conditionsAβ1-42can aggregated into the maximum after24h incubation.Then in order to further validate the accuracy of using Acumen to detect the Th-T fluorescence, we use the metal chelator clioquinol (CQ) which was known to bethe inhibitor of Zn2+induced Aβ1-42aggregation as a positive compound to influencethe aggregation of Aβ1-42. Furthermore in the experiment some impurity which canbind to Th-T and has strong fluorescence at485nm can influence the intensity offluorescence, but it can’t be precluded when using micro reader to detection. But wecan eliminate fluorescence intensity of impurities by limiting the experimentparameters and reduce the influence to the total intensity of Aβ1-42. So after all thestudy done above we can conclude that Acumen can be used to detect fluorescencequantitative together with image observation.Conclusion: a new way to detect the aggregation of Aβ with high speed and onthe basis of picrure viewing had been established.Section ⅡThe screening of compounds on Aβ1-42aggregation and Zn2+inducedaggregation ofAβ1-42and futher evaluation at molecule levelThe aggregation of Aβ1-42can be self-assembed and can also be induced bymetal. So in this section we used the metod established in part Ⅰ to screen19compounds (40μM for monomer or40μg/mL for mixture)in the hope of gettingsome compounds which have the activity of influence the aggregation of Aβ1-42(10μM). CQ was chosen as the positive control. After the screening some evaluationswere done to find how the active compounds influence the aggregation of Aβ1-42.1. Screening of compoundsThe screening of HYF series compounds (40μM)(from the pharmaceuticalchemistry laboratory) showed that all the compounds except for LL4-3-Ⅱ can couldsignificantly inhibit the monomer Aβ1-42from aggregation and also Zn2+(20μM)induced Aβ1-42aggregation, when the compounds were incubated together withmonomer Aβ1-42for24h. However LL4-3-Ⅱ can significantly inhibit Zn2+inducedAβ1-42aggregation. Results of nature product screening showed that the C1900, BSG,JD-30, A102could significantly inhibit monomer Aβ1-42aggregation or Zn2+(20μM)inducedAβ1-42aggregation.The image results showed that CQ, HYF118C and C1900can reduce the numberof aggregated Aβ1-42both under Acumen and under the fluorescence microscope,furthermore under the same condition CQ, HYF118C and C1900can obviouslyweake the fluorescence. F2. Exclude of false positive resultsIn order to eliminate the possible false positive results, the absorption spectrum(300nm to600nm) of compounds were scanned，the results showed that only A102had strong absorption at485nm. So we can conclude that CQ, HYF118C and C1900 can’t cause a false positive result for their spectroscopy properties.Furthermore a competitive binding assay was applied to determine whether Th-Tand CQ have the same binding site on Aβ1-42. The Competitive binding assay showedthat CQ, HYF118C and C1900and Th-T did not bind the same site onAβ1-42.Conclusion: we have got HYF118C and C1900which can resist the aggregationof Aβ1-42through the screening and exclusion of false positive result together withprior study in our laboratory. So in the following part we just do the research onHYF118C and C1900.3. The molecule evaluation of compounds3.1Dose response effect of CQ, HYF118C and C1900onAβIn order to know more about the effect of the three compounds on Aβ1-42, thedose response study were done. The results showed that when incubated withmonomer Aβ1-42, CQ could inhibit the aggregation of Aβ1-42in a dose dependentmanner, the IC50were6.1μM (without Zn2+) and4.3μM (with Zn2+), in the sameway HYF118C can also decrease the influence intensity in a dose dependent manner,the IC50were22.4μM (without Zn2+) and18.6μM (with Zn2+), and the results ofC1900showed that C1900can reduce the influence intensity in a dose dependentmanner, the IC50were14.8μM (without Zn2+) and17.3μM (with Zn2+), so we canconclude that CQ, HYF118C and C1900can inhibit the aggregation of Aβ and theaggregation induced by Zn2+directly in vitro. Zn2+has little influence on the effect.The inhibition capability of HYF118C及C1900were weaker than CQ.However when incubated with aggregated Aβ1-42, CQ decreased the influenceintensity in a dose dependent manner, the IC50were7.5μM (without Zn2+) and6.1μM (with Zn2+), for HYF118C the IC50were18.7μM (without Zn2+) and14.7μM(with Zn2+), and for C1900the IC50were8.7μM (without Zn2+) and9.5μM (withZn2+). So we can conclude that CQ, HYF118C and C1900can depolymerize theaggregated Aβ and induced aggregation of Aβ1-42directly in vitro. Zn2+has littleinfluence on the effect. Moreover the depolymerize capability of HYF118C andC1900were weaker than CQ, too.3.2The change of secondary structure ofAβ by circular dichroism spectra (CD)In the study Circular dichroism spectra (CD) was used to detect β-sheetformation of Aβ1-42. CD spectra showed that the β-sheet content of Aβ1-42was in goodlinner relationship with the concentrition of Aβ1-42, and after incubated for24h theincrease of β-sheet during the incubation reach the plateau phase. Moreover theinfluence of CQ, HYF118C and C1900(240μM) on60μM Aβ1-42showed only CQcan decrease the content of β-sheet, but HYF118C and C19008C had little influenceon β-sheet ofAβ1-42. 3.3The change of Aβ by the active compounds by transmission electronmicroscope (TEM)CQ, HYF118C and C1900(40μM) can obviously change the formation of Aβ1-42(40μM), and there was less conjuctions between the molecules.Conclusion: CQ, HYF118C and C1900can inhibit the aggregation of Aβ1-42andcan also depolymerize aggregated Aβ1-42in dose response manner, and Zn2+had littleinfluence on the effect. CQ could decrease the β-sheet content of Aβ1-42. CQ,HYF118C and C1900(40μM) can obviously change the formation of Aβ1-42, and aftertreated by the compounds there was less conjuctions between the molecules.Section Ⅲ The evaluation of CQ, HYF118C and C1900CQ, HYF118C and C1900have been known as the inhibitors of Aβ1-42aggreation, so in order to find wheather the compounds have bioactivities related toAβ. The evaluations were done on Aβ1-42-induced cytotoxicity model, APP/PS1miceand SAMP8mice.1. The effect of CQ, HYF118C and C1900onAβ1-42induced cytotoxicityCCK-8assay was performed to examine the cell viablity. And the stateobservation was done under the micro scop.First, the effects of CQ, HYF118C, C1900on SH-SY5Y showed that only10μMCQ had little cytotoxic effect, when the concentrition were lower than10μMHYF118C and C1900had no harm to the cells.Then, the effects of CQ, HYF118C, C1900of40μM Aβ1-42induced injure onSH-SY5Y cells showed when the concentrition reach40μM the cell survival wasabout50%, and CQ, HYF118C, C1900can effectively improce the cell viability.Morover, the state observation results showed40μM Aβ1-42can significantly causedamage to the cells compared to the normal group, however the three compounds cansignificantly improve the state.The above investigation indicate that CQ, HYF118C, C1900had neuroprotectiveeffect againstAβ1-42-induced cell damage.2. The effect of CQ, HYF118C and C1900on animals2.1The effect of CQ, C1900on behavior and the synaptic plasticity of APP/PS1mice13-14months APP/PS1mice were chosen to evaluate the effect of CQ, andC1900. The mice received an oral adminstration for11days with the dose of CQ30mg/kg/d, C1900100mg/kg/d. And nest construction test, locomotor activities, objectrecognition test, step down test, Morris Water Maze, long-term potentiation (LTP)were done to investigate the effect of compounds on the social behavior, memory andsynaptic plasticity. Results showed no change on the weight of the mice, C1900could significantlyimprove the nest-construction behavior compared to the APP/PS1model group,could improve the escape lantency in the probe test of Morris Water Maze, LTPresults showed C1900could significantly shift the―Input-Output‖(I-O) curve to left,and could improve the population spike (PS), and this indicate that C1900canimprove the synaptic plasticityof APP/PS1mice. However, CQ could only improvethe escape lantency in the probe test of Morris Water Maze compared to the modelgroup.2.2The effect of CQ, HYF118C, C1900on behavior and the synaptic plasticity ofSAMP8miceFurthermore our study also investigate the effect of CQ (30mg/kg/d), HYF118C(30mg/kg/d), C1900(100mg/kg/d) on SAMP8mice. The results showed the threecompounds had no obviously effect on the behavior and the synaptic plasticity ofSAMP8mice.Conclusion:The above investigation indicate that HYF118C, C1900and CQ hadthe neuroprotective effect on Aβ1-42-induced cell damage. The animals test showedC1900could improve the nest behavior, memory and synaptic plasticity of APP/PS1mice and could be a potential drug for AD treatment.Section Ⅳ Conclusion1. In the study a high flux, rapid, direct-viewing method was established2. HYF118C and C1900were found to be the inhibitors of Aβ1-42aggregation, andZn2+could not influence the effect, CQ can inhibit the β-sheet of Aβ1-42.3. HYF118C and C1900had the neuroprotective effect against Aβ1-42-induced celldamage.4. C1900could improve the synaptic plasticity of both APP/PS1and SAMP8mice,could improve the nesting behavior of APP/PS1mice, could also improvememory of APP/PS1mice, and the effect of C1900was better than CQ.