| PartⅠInterfering with protein-protein interactions or protein-nucleic acid interactions have been regarded as daunting goals in drug discovery. Major strides have been made the last few decades in developing small molecule agents to target protein-protein interactions. However, regulation of protein-RNA interactions lags behind, primarily due to the fact that RNA molecules pose a particular challenge with their high flexibility. RNA-binding proteins (RBPs) play key roles in post-transcriptional control of RNAs, which, along with transcriptional regulation, is a main method of regulating patterns of gene expression during development.In this part, we focus on the identification of molecular probes that interfere with the dsRNA binding region of TLR3as a demonstration of using highly specific and selective small molecule agents to target the protein-RNA interface. It contained:(1) Define the targeting site on TLR3according the crystal structure of TLR3/dsRNA complex.(2) All1.2million compounds were first docked and ranked using High Throughput Virtual Screening (HTVS) by Glide5.6, continued with standard precision (SP) Glide for the top10000compounds. The resultant top5000compounds were then docked using the more accurate and computationally intensive extra-precision (XP) mode. Initial top-ranked100compounds were selected out. The resulting100candidates were subsequently filtered by reasonable chemical structure, interaction with the residues, molecule weight and binding energy. Consequently,9potential TLR3inhibitors were designated and purchased for vitro assaying.(3) According the vitro assay results, we get2mother compounds.(4) With the lead structures identified, we developed a concise synthetic route for both leads, which allows an extensive structure-activity relationship (SAR) analysis. Various substitutions with different size and electron withdrawing/donating capability were examined on the aromatic systems, totally72new compounds. To inspect the impact on the activities imposed by the stereogenic center, both R-and S-isomers were prepared.(5) We carried out the downstream signaling evaluation for the best inhibitory activity compound in virto, including TNF-α, IL-1β and IFN-β. We also test it’s toxicity to P450enzyme and a panel of representative kinases.Research result demonstrated:(1) Two compounds (T5626448and T5260630) showed mild inhibitory activities in whole cells, with IC50values of154±6μM and145±4μM, respectively. Both of these two compounds are derivatives of D-phenylalanine, suggesting the D-phenylalanine backbone as the scaffold to develop small molecule inhibitors of TLR3.(2) Substituting the7-membered ring (T5626448) with a phenyl group, introduce α-F on the phenyl group and a-Cl on the thiophene rings increased the inhibitory activity about45fold (4a IC503.44±0.41μM).(3) With the absence of either-F or-Cl, the inhibitory activity is greatly decreased, even vanish.(4) These T5626448derivatives’inhibitory effects are stereo-dependent, with the R-enantiomers generally demonstrating higher potency than the S-enantiomers.(5) An additional hydroxyl group greatly decreases the activity (7a vs4a), suggesting higher hydrophobicity is favored at the amino acid side chain.(6) Dose dependent inhibitory activity test shows that4a inhibibit all the NO signal at the concentrate of40μM.(7) We found that4a inhibits TLR3signaling without affecting other TLRs, showing it is highly selective in intact cells.(8) Further, compound4a was found to have low cytotoxicity. CYP450tests showed that4a did not affect a panel of cytochrome enzymes (Cyp3A4,2D6,2C19and1A2). The low toxicity of4a was further confirmed in RAW264.7cells using the established WST-1methodology.(9) Compounds4a was profiled against a panel of12representative kinases (AKT1, CAMK1, DDR2, GSK-3α, MAPK1, MET, PAK1, PDGFRB, PIM1, PKC-γ, PLK4and SRC) using the KinaseSeekerTM assay. After it was determined that compounds4a did not inhibit the luciferase control, profiling was done in duplicate against each kinase.(10) Biophysical tests were carried out for4a, along with the negative control compound la, to demonstrate that4a directly binds to TLR3. Fluorescence anisotropy assays showed that4a competes with dsRNA for binding to TLR3with a Kj of2.96±0.32μM. Increasing4a’s concentration to68μM decreased the anisotropy to background levels. These data were then fitted to a one-site-competition model.(11) Lastly, we used a secondary cellular assay to confirm that4a also inhibits the downstream signaling transduction mediated by the formation of the TLR3/dsRNA complex. In addition to TLR3signaling suppression, the release of the proinflammatory cytokines, TNF-α, IL-1β and IFN-β, were investigated. Results shown in Figure4further confirmed that compound4a suppresses TLR3-mediated inflammation response, such as TNF-α, IL-1β and IFN-β.We also investigated, synthesized and tested compounds as TLR4inhibitors. Scientist reported that TLR4plays an essential role in microglial activation that contributes to the development of morphine tolerance and thereby compromises the analgesic effects of morphine. This exciting discovery provides a novel avenue for therapeutic development to attenuate morphine tolerance by blocking TLR4signal transduction in glial cells. Our group has reported the lead compound as TLR4inhibitor in the previous research. In this thesis we focus on decrease the toxicity and increase the rigid of the lead compound. We designed and synthesized5linear and2macrocyclic compounds, and the result showed that the linear structure increased the inhibitory active and the macrocyclic decreased the toxicity. Compound17can inhibit50%of the LPS-induced cytokine relase in human whole blood at50μM, such as IL-1β, IL-6, IL-8and TNF-α, which demonstrate it can be further optimization.Part ⅡMulti-drug-resistant Gram-positive and Gram-negative gens have become a serious problem in hospitals and the community. Particularly alarming is the emergence of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE) and vancomycin-resistant Enteroccocus faecium (VRE). Drug discovery efforts have been significantly intensified in the past years to search for more effective antibacterial agents with a broader spectrum of activity and especially activity against resistant pathogens to fight infectious diseases.With the developing of molecule biology tetechology, define a drug target for development of antibacterial drug is becoming promising. Bacterial type Ⅱ fatty-acid synthesis (FASⅡ) is an attractive target for antibacterial drug discovery. The initiation condensing enzyme, Escherichia coli β-Ketoacyl-acyl carrier protein synthase Ⅲ (ecKAS Ⅲ, FabH), and elongation condensing enzymes, KAS Ⅰ/Ⅱ, are essential components of fatty-acid biosynthesis and are highly conserved among key pathogens. Here, we synthesized64peptide&Schiff bases (PSB) as FabH inhibitor and the inhibitory activities against FabH were investigated in vitro. Top15PSB compounds which posses good FabH inhibitory activity (low IC50) were picked out to test their antibacterial activities against two Gram-negative bacterial strains(Escherichia coli ATCC35218and Pseudomonas aeruginosa ATCC13525) and two Gram-positive bacterial strains (Bacillus subtil is ATCC6633and Staphylococcus aureus ATCC6538), expecting to exploit potent antibacterial agent with broad-spectrum antibiotics activity. As expected, the results demonstrate that the antimicrobial compound,2d,5f,7e and5e, can be as an inhibitor of FabH and as a potential antibiotics agent, displaying MIC values in the range0.39-6.25μg/mL against various bacteria. The AutoDock study for the inhibitors docked into the active sites of FabH has also been carried out. Compounds5f,7e and5e can bind into the mouth of the substrate tunnel by interaction with Gly209and Gly152. |
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