免疫蛋白TLR3和4的抑制剂的设计、合成、结构修饰及信号传导研究
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摘要
上篇：
干扰、调节蛋白质与蛋白质,或蛋白质与核酸之间的相互作用是当今药物研发的重要靶点。从过去的几十年到现在,主要的研究都聚焦在利用小分子来调节蛋白质与蛋白质之间的相互作用,并且已取得了巨大的进展。而研究蛋白质与核酸(RNA)之间的相互作用却相对滞后,一个很重要的原因是RNA具有很好的柔韧性、灵活性,与蛋白紧密结合,不易受到外界干扰。同时,RNA结合蛋白(RBPs)对转录后调控RNA有重要的影响,也对进化中的基因表达模式起到了关键的作用。
上篇主要工作集中在探寻小分子抑制剂,来干扰RNA与免疫蛋白TLR3之间的相互作用,从而抑制TLR3的信号产生,达到治疗炎症性疾病的目的。(1)通过TLR3/dsRNA复合物的晶体结构(PDB:3CIY),定义出小分子与TLR3之间的相互作用靶点。(2)运用软件Glide5.6,借助高通量计算机筛选(High Throughput Virtual Screening, HTVS)的办法,从120万个药物小分子数据库中筛选出10000个有可能的小分子抑制剂；接下来对这10000个小分子进行标准精度筛选(standard precision, SP),得到了5000个更有可能的小分子抑制剂；紧接着进行高精确度筛选(extra-precision, XP)得到了100个潜在的小分子抑制剂；然后根据结构的合理性、与活性残基作用的能力、分子量、以及结合能的大小,挑选出了9个化合物,向Enamine购买。(3)对这9个化合物进行体外抑制TLR3的活性评价,得到了2个母体化合物。(4)随后以这2个母体化合物为骨架,考虑到电子等排体效应、电负性效应、空间异构效应等,展开了结构修饰和优化,共设计、合成出了72个新化合物,并对其进行了构效关系(Structure-activity Relationship, SAR)研究。(5)对抑制效果最好的潜在药物,进行了下游信号传导——肿瘤坏死因子α(TNF-α)、白细胞介素1p(IL-1β)、干扰素β (IFN-β)的研究；同时也对细胞色素P450酶和常见的激酶进行了毒性及抑制活性评价。
研究结果显示出：(1)2个母体化合物(T5626488和T5260630)都含有苯丙氨酸(Phe)的结构,对TLR3的抑制活性分别为IC50154±6μM和145±4μM。(2)当用苯环代替T5626488中的7元环,并在苯环上引入F、五元环上引入Cl后(4a),其对TLR3抑制活性提高了45倍,达到最好(IC503.44±0.41μM)。(3)F与Cl二者缺一,抑制活性将大幅减少,甚至消失(9a-11a,13a)。(4)在有抑制活性(IC50<100μM)的手性对应异构体中,R构型均好于对应的L构型。(5)用多一个羟基的酪氨酸(7a,7b)代替苯丙氨酸(4a,4b)之后,无论是R还是S构型,其抑制TLR3的活性都大大降低。(6)不同浓度的抑制活性测试显示4a在40μM左右已经将TLR3的信号全部抑制。(7)特异性测试显示,4a只针对TLR3有较强的抑制活性,而对TLR1/2, TLR2/6, TLR4,和TLR7没有抑制活性。(8)WST针对Raw264.7Cell细胞毒性测试显示4a在81μM时细胞没有毒性；在细胞色素P450酶家族(Cyp3A4, Cyp2D6, Cyp2C19和Cyp1A2)毒性测试进一步显示,相对于特定的P450酶家族的抑制剂而言,4a在25和50μM时,毒性均没有超过特定的阳性对照抑制剂。(9)对激酶家族中有代表性的的12种激酶测试显示,4a在10μM时对所有激酶都仍然保持着100%的抑制活性。(10)生物物理实验部分：通过荧光淬灭滴定(Fluorescence Quenching Titration)验证4a与TLR3相结合。通过荧光各向异性测定(Fluorescence Anisotropy)显示4a能够与dsRNA竞争来与TLR3相结合；在浓度为68μM时,几乎将所有的dsRNA都竞争出局；归一化结果显示,4a与dsRNA竞争结合TLR3符合单点竞争结合模型(one-site-competition model)。(11)下游信号传导研究显示进一步证明,4a能够通过与TLR3相互作用,有效地干扰TLR3与dsRNA相结合,抑制了下游信号因子TNF-α、IL-1β和IFN-β的产生。
上篇也对TLR4的抑制剂的合成进行了研究。文献报导,TLR4与吗啡的治疗疼痛作用有关,如果抑制TLR4的产生,用少量的吗啡就可以达到同样的止疼效果。之前课题组已经报导了TLR4的抑制剂的筛选、合成和活性研究。本论文里的主要工作是进一步降低先导化合物的毒性和提高先导化合物结构的刚性,共设计合成了5个直链和2个大环的抑制剂。结果显示,直链的2个化合物抑制活性有所提高,大环的化合物毒性有所降低。血液内释放的细胞因子的测试结果显示,不同浓度下17对血液里的IL-1β, IL-6, IL-8和TNF-α的抑制效果非常显著,在50μM达到最佳,值得进一步深入研究。
下篇：
革兰氏阳性和阴性的病原菌对药物的耐药性的不断出现,给医院的用药和公共的卫生安全带来了极大地威胁。尤其令人震惊的是出现了耐甲氧西林金黄色葡萄球菌(MRSA)、耐表皮葡萄球菌(MRSE)和耐万古霉素的Enteroccocus faecium菌的出现,迫使我们不得不加快步伐开发新型的抗菌药或进一步提高现有药物的抗菌活性。
随着分子生物学技术的进一步发展,科学家通过确定抗菌药物的靶点来进行抗菌药物研究。近年来,细菌脂肪酸生物合成途径中所涉及的关键酶引起了研究人员的广泛兴趣。其中β-酮脂酰-ACP合成酶Ⅲ(β-Ketoacyl-ACP synthase Ⅲ, FabH, KAS Ⅲ)控制着细菌脂肪酸生物合成的起始步骤,是细菌脂肪酸生物合成的关键酶,是最有望成为新型抗菌药物靶标之一。在这里,我们合成了64个酰胺希夫碱类化合物,测定了它们对FabH的抑制作用。对其中抑制效果较好的前15个化合物进行了抗革兰氏阳性菌(芽孢杆菌Bacillus subtilis ATCC6633,金黄色葡萄球菌Staphylococcus aureus ATCC6538)和革兰氏阴性菌的测试(大肠杆菌Escherichia coli ATCC35218,假单胞菌Pseudomonas aeruginosa ATCC13525),结果显示2d、5f、7e和5e对这4种菌都有较好的抑制作用,MIC在0.39-6.25之间。为了更好的研究构效关系和抑制机理,我们采用分子模拟的方法,选取FabH的晶体结构(1HNJ.pdb),将对FabH有较好抑制活性的分子进行了模拟研究。化合物5f、7e和5e中的亚胺基氢、酚羟基氢与FabH中的活性残基Gly152和Gly209存在分之间的氢键相互作用。这些分子模拟数据为进一步的机制研究提供了思路和方向。
PartⅠ
Interfering 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 Ⅱ
Multi-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.
干扰、调节蛋白质与蛋白质,或蛋白质与核酸之间的相互作用是当今药物研发的重要靶点。从过去的几十年到现在,主要的研究都聚焦在利用小分子来调节蛋白质与蛋白质之间的相互作用,并且已取得了巨大的进展。而研究蛋白质与核酸(RNA)之间的相互作用却相对滞后,一个很重要的原因是RNA具有很好的柔韧性、灵活性,与蛋白紧密结合,不易受到外界干扰。同时,RNA结合蛋白(RBPs)对转录后调控RNA有重要的影响,也对进化中的基因表达模式起到了关键的作用。
上篇主要工作集中在探寻小分子抑制剂,来干扰RNA与免疫蛋白TLR3之间的相互作用,从而抑制TLR3的信号产生,达到治疗炎症性疾病的目的。(1)通过TLR3/dsRNA复合物的晶体结构(PDB:3CIY),定义出小分子与TLR3之间的相互作用靶点。(2)运用软件Glide5.6,借助高通量计算机筛选(High Throughput Virtual Screening, HTVS)的办法,从120万个药物小分子数据库中筛选出10000个有可能的小分子抑制剂；接下来对这10000个小分子进行标准精度筛选(standard precision, SP),得到了5000个更有可能的小分子抑制剂；紧接着进行高精确度筛选(extra-precision, XP)得到了100个潜在的小分子抑制剂；然后根据结构的合理性、与活性残基作用的能力、分子量、以及结合能的大小,挑选出了9个化合物,向Enamine购买。(3)对这9个化合物进行体外抑制TLR3的活性评价,得到了2个母体化合物。(4)随后以这2个母体化合物为骨架,考虑到电子等排体效应、电负性效应、空间异构效应等,展开了结构修饰和优化,共设计、合成出了72个新化合物,并对其进行了构效关系(Structure-activity Relationship, SAR)研究。(5)对抑制效果最好的潜在药物,进行了下游信号传导——肿瘤坏死因子α(TNF-α)、白细胞介素1p(IL-1β)、干扰素β (IFN-β)的研究；同时也对细胞色素P450酶和常见的激酶进行了毒性及抑制活性评价。
研究结果显示出：(1)2个母体化合物(T5626488和T5260630)都含有苯丙氨酸(Phe)的结构,对TLR3的抑制活性分别为IC50154±6μM和145±4μM。(2)当用苯环代替T5626488中的7元环,并在苯环上引入F、五元环上引入Cl后(4a),其对TLR3抑制活性提高了45倍,达到最好(IC503.44±0.41μM)。(3)F与Cl二者缺一,抑制活性将大幅减少,甚至消失(9a-11a,13a)。(4)在有抑制活性(IC50<100μM)的手性对应异构体中,R构型均好于对应的L构型。(5)用多一个羟基的酪氨酸(7a,7b)代替苯丙氨酸(4a,4b)之后,无论是R还是S构型,其抑制TLR3的活性都大大降低。(6)不同浓度的抑制活性测试显示4a在40μM左右已经将TLR3的信号全部抑制。(7)特异性测试显示,4a只针对TLR3有较强的抑制活性,而对TLR1/2, TLR2/6, TLR4,和TLR7没有抑制活性。(8)WST针对Raw264.7Cell细胞毒性测试显示4a在81μM时细胞没有毒性；在细胞色素P450酶家族(Cyp3A4, Cyp2D6, Cyp2C19和Cyp1A2)毒性测试进一步显示,相对于特定的P450酶家族的抑制剂而言,4a在25和50μM时,毒性均没有超过特定的阳性对照抑制剂。(9)对激酶家族中有代表性的的12种激酶测试显示,4a在10μM时对所有激酶都仍然保持着100%的抑制活性。(10)生物物理实验部分：通过荧光淬灭滴定(Fluorescence Quenching Titration)验证4a与TLR3相结合。通过荧光各向异性测定(Fluorescence Anisotropy)显示4a能够与dsRNA竞争来与TLR3相结合；在浓度为68μM时,几乎将所有的dsRNA都竞争出局；归一化结果显示,4a与dsRNA竞争结合TLR3符合单点竞争结合模型(one-site-competition model)。(11)下游信号传导研究显示进一步证明,4a能够通过与TLR3相互作用,有效地干扰TLR3与dsRNA相结合,抑制了下游信号因子TNF-α、IL-1β和IFN-β的产生。
上篇也对TLR4的抑制剂的合成进行了研究。文献报导,TLR4与吗啡的治疗疼痛作用有关,如果抑制TLR4的产生,用少量的吗啡就可以达到同样的止疼效果。之前课题组已经报导了TLR4的抑制剂的筛选、合成和活性研究。本论文里的主要工作是进一步降低先导化合物的毒性和提高先导化合物结构的刚性,共设计合成了5个直链和2个大环的抑制剂。结果显示,直链的2个化合物抑制活性有所提高,大环的化合物毒性有所降低。血液内释放的细胞因子的测试结果显示,不同浓度下17对血液里的IL-1β, IL-6, IL-8和TNF-α的抑制效果非常显著,在50μM达到最佳,值得进一步深入研究。
下篇：
革兰氏阳性和阴性的病原菌对药物的耐药性的不断出现,给医院的用药和公共的卫生安全带来了极大地威胁。尤其令人震惊的是出现了耐甲氧西林金黄色葡萄球菌(MRSA)、耐表皮葡萄球菌(MRSE)和耐万古霉素的Enteroccocus faecium菌的出现,迫使我们不得不加快步伐开发新型的抗菌药或进一步提高现有药物的抗菌活性。
随着分子生物学技术的进一步发展,科学家通过确定抗菌药物的靶点来进行抗菌药物研究。近年来,细菌脂肪酸生物合成途径中所涉及的关键酶引起了研究人员的广泛兴趣。其中β-酮脂酰-ACP合成酶Ⅲ(β-Ketoacyl-ACP synthase Ⅲ, FabH, KAS Ⅲ)控制着细菌脂肪酸生物合成的起始步骤,是细菌脂肪酸生物合成的关键酶,是最有望成为新型抗菌药物靶标之一。在这里,我们合成了64个酰胺希夫碱类化合物,测定了它们对FabH的抑制作用。对其中抑制效果较好的前15个化合物进行了抗革兰氏阳性菌(芽孢杆菌Bacillus subtilis ATCC6633,金黄色葡萄球菌Staphylococcus aureus ATCC6538)和革兰氏阴性菌的测试(大肠杆菌Escherichia coli ATCC35218,假单胞菌Pseudomonas aeruginosa ATCC13525),结果显示2d、5f、7e和5e对这4种菌都有较好的抑制作用,MIC在0.39-6.25之间。为了更好的研究构效关系和抑制机理,我们采用分子模拟的方法,选取FabH的晶体结构(1HNJ.pdb),将对FabH有较好抑制活性的分子进行了模拟研究。化合物5f、7e和5e中的亚胺基氢、酚羟基氢与FabH中的活性残基Gly152和Gly209存在分之间的氢键相互作用。这些分子模拟数据为进一步的机制研究提供了思路和方向。
PartⅠ
Interfering 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 Ⅱ
Multi-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.
引文
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2. Glide, version 5.6, Schrodinger, LLC, New York, NY,2010.
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3. Manning, G.; Whyte, D.; Martinez, R.; Hunter, T.; Sudarsanam, S. Science 2002, 298,1912-1934.
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