Design, Synthesis And Biological Evaluation Of Compounds Regulating The TLRs/MyD88 Signaling Pathway

Alzheimer's disease (AD) is a progressive multifaceted neurodegenerative disorder accompanied with cognitive impairment, memory loss and abnormal behavior. The pathologic hallmarks of AD are the presence of senile plaques (SPs) and neurofibrillary tangles (NFTs) in the brain. The traditional drug design strategy was focused on'one-target, one-disease'in many recent researches has not met with the expected success for AD. Efficient therapy is more likely to be achieved by drugs that could intervene to multiple targets and node proteins of AD, simultaneously regulate the related targets of the entire AD etiology network.Regulating the TLRs/MyD88 signaling pathway of the innate immune system would have an impact on the related targets of the entire AD etiology network and could increase the clearance to A?, keep the balance of A? in brain and reduce the aggregation of A?. Consequently, it could attenuate the symptom of AD. The aim of this work is to design a series of novel non-peptidomimetic compounds which acted on adaptor protein MyD88 through the computer-aided drug design method, and provide the necessary theoretical basis and experimental evidence to prenvent the pathogenesis of AD by regulating the innate immune systerm.The main contents and results are listed as follows:1. Design of compounds acting on the key molecule MyD88 in TLRs signaling pathwayCombined with the 3D crystal structure of MyD88, the action mechanism of MyD88 (dimerization) and the effect that acted on MyD88 by ST2825 (a peptidomimetic inhibitor), we used the software Catalyst to generate the optimal pharmacophore model. On the basis of the constructed pharmacophore model, we screened the library to find structures (2-piperidone derivatives and 2-aminothiazole derivatives) that matched this model well and also had good estimated activities. The docking study results showed that the compounds could change the configuration and the domain interface electron cloud density of MyD88, and also affect the homodimerization or heterdimerization of MyD88 and TLR signaling pathway.2. Synthesis and structural dissection of target compoundsAccording to the structure characteristics of the designed compounds, the best synthetic route was designed and 46 compounds in two classes were obtained. All the compounds were confirmed and analyzed by UV, IR,1H-NMR,13C-NMR, MS and HPLC.3. Biological evaluation of target compounds3.1 Inhibition of self-mediated A?1-2 aggregationThe results of ThT fluorescence assay showed that the bulk of the target compounds possessed moderate to good potencies of A?1-42 aggregation inhibition in a concentration-dependent manner relative to that of curcumin. The aminothiazole compound P2 displayed the highest potency (84.48±0.41%at 20?M). The results of AFM assay manifested that the difference of the morphologies were mainly contributed to the strong interaction between AP1-42 aggregates and the compound. Obviously, the compound could effectively inhibit A?1-42 fibrillogenesis.3.2 Effect of compounds on the production of pro-inflammatory cytokinesMost of the compounds could effectively inhibit the production of several different pro-inflammatory cytokines stimulated by LPS in microglia. Thus, it could ameliorate the local inflammation atmosphere and regulate the pro-inflammatory cytokines in a rational level. Compound L15 could effectively reduce the production of these three cytokines (TNF-?, IL-1? and IL-6) with an IC50 value of 0.08±0.02 ?M,0.21 ± 0.06 ?M and 0.03± 0.01?M, respectively.3.3 Assays for cytoprotective action in SH-SY5Y cellsIn these assays, most of the 2-aminothiazole derivatives showed moderate to good potencies to protect SH-SY5Y cells in inflammation mediated neurotoxicity-induced, H2O2-induced and A?-induced injury models. While, the 2-piperidone derivatives could only protect SH-SY5Y cells in inflammation mediated toxicity-induced injury model. Among these compounds, P2 showed highly protective (EC500.1858 ?M in H2O2-induced; 79.22±3.22% at 10 ?M in A?-induced; 81.88% at 5?M in inflammation mediated neurotoxicity-induced, respectively).3.4 Co-IP assayThe Co-IP assay directly supported a strong interaction of the compounds with MyD88. Compounds could inhibit MyD88 homodimerization in a dose-dependent manner.In conclusion, most of the designed compounds showed multifunctional properties by regulating the TLRs/MyD88 signaling pathway and affecting the multiple relative targets of the entire AD etiology network. Consequently, these compounds emerged as promising candidates for further development of novel multifunctional agents for AD treatment.The characters and innovation points:The strategy of developing AD medication through regulating the TLRs/MyD88 signaling pathway of the innate immune system and modulating the related targets of the entire AD etiology network provides a new idea and approach for the prevention and treatment of AD. The two classes of designed compounds were first obtained to emerge as the non-peptidomimetic compounds regulating the TLRs/MyD88 signaling pathway.