钴胺素依赖的蛋氨酸合酶催化N5-甲基四氢叶酸转移甲基至同型半胱氨酸生成蛋氨酸和四氢叶酸,直接参与蛋氨酸循环、叶酸循环及含硫氨基酸代谢,与DNA、蛋白质合成及生物甲基化有密切关系。本研究采用蛋白层析技术,将大鼠肝匀浆经超声破碎和高速离心处理后,依次经过DE-52批处理、Q Sepharose Fast Flow离子交换层析和CHT陶瓷羟基磷灰石吸附柱层析进行纯化,并对纯化产物进行了SDS-PAGE和Western blotting鉴定。采用分光光度法测定蛋氨酸合酶的活性,对纯化酶的酶促反应动力学进行了研究,确定了最佳反应条件,动力学结果显示蛋氨酸合酶的双底物酶促反应的机制为乒乓机制。研究表明,采用层析技术纯化得到的蛋氨酸合酶适用于以其为靶点的化合物高通量筛选。
trans-3-Isopropyl-4-（2-methylcyclohexyloxy）-6-phenethylpyridin-2（1H）-one, as reverse transcriptase （NNRTIs）, exhibited significant potent activity not only against wild-type HIV-1 strains but also on mutant strains. For furthering study this compound, the original synthetic route should be shorten to improve the total yield. In this report, we designed an efficient synthetic strategy to obtain the target compound with higher yield.
Among the structurally diverse NNRTIs, pyridinone scaffolds demonstrate high potency against HIV-1 wild type and drug-resistant strains. During the optimization of our pyridinone compound 1（LAM-trans）, we found that the introduction of the N atoms in the C-4 position could dramatically improve the water solubility（7b）, whereas protonation of the piperidine N atom resulted in a decrease in its hydrophobic interaction with the binding pocket. In particular, protonation altered the orientation of the alicyclic rings in the hydrophobic pocket, thus impeding the formation of key halogen bond and eventually leading to a huge change in anti-HIV-1 RT activity. These results provided theoretical and experimental basis for the subsequent structural modification of pyridinone compounds.
Yunqi LiuXixi LiXiaodong DouChao TianZhili ZhangJunyi LiuXiaowei Wang
HIV-1 reverse transcriptase（RT） inhibitors are major components of HAART（highly active antiviral therapy）. The S-DABOs（dihydro-alkylthio-benzyl-oxopyrimidines） series and their similar skeletons have exhibited preferable activities to inhibit HIV-1 RT. In the present study, we generated field-based QSAR models using common structure alignment, which was characterized by Gaussian steric, electrostatic, hydrophobic, hydrogen bond donor, hydrogen bond acceptor and aromatic ring fields（R2 = 0.8421, RCV2 = 0.5949 for the training set, Q2 = 0.5486, Pearson-r = 0.7460 for the test set）. Docking, pocket surface and contour map analyses were carried out. Key pharmacophore features were investigated, including（i） π-π interaction with residue Tyr181, Tyr188 and Trp229, σ-π interaction with His236,（ii） hydrogen bond with residue Lys101 and halogen bond with residue Tyr188. The docking analysis and field-based QSAR models could provide reasonable guidance in the rational design of potent HIV-1 RT inhibitors.
目的:改进经典抗叶酸类药物关键中间体4-氨基-5-甲酰基-8,10-二去氮杂四氢叶酸二乙酯侧链的水解条件。方法:以经典叶酸拮抗剂侧链N-(4-氨基苯甲酰)-L-谷氨酸二乙酯(1)为反应原料,尝试了氢氧化钠(Na OH)和氢氧化钾(KOH)2种碱催化、20~180 min 5种反应时间和0.175~1 mol/L 3种碱浓度的反应条件,用高效液相色谱法检测目标产物和副产物,最终确定副产物为单酯水解产物以及酰胺键水解产物,并以此为依据完成了4-氨基-5-甲酰基-8,10-二去氮杂四氢叶酸二乙酯(5)水解条件的优化。结果:改进后的4-氨基-5-甲酰基-8,10-二去氮杂四氢叶酸二乙酯侧链的水解条件为0.3 mol/L KOH溶液中室温条件反应60 min,在该反应条件下,水解反应收率为95.6%。反应产物通过磁共振氢谱(1H nuclear magnetic resonance,1H NMR)、磁共振碳谱(13C nuclear magnetic resonance,13C NMR)和电喷雾飞行时间质谱(electrospray ionization time of flight mass spectrometry,ESI-MS)分析鉴定后结构正确,并通过高效液相色谱法确定其纯度为96%。新的水解反应条件避免了副产物的生成,提高了反应收率。结论:利用新的水解条件可以简便、高效地完成4-氨基-5-甲酰基-8,10-二去氮杂四氢叶酸二乙酯的水解反应,该条件对经典叶酸拮抗剂的合成和生产工艺的改进也有重要的意义。
Aziridine derivatives of N^5-methyltetrahydrofolate were designed and synthesized based on the mechanism of methionine synthase, and their biological activities were investigated as well. The aziridine derivatives 1 and 6 exhibited superior inhibitory activities（IC50：5.05 and 4.15 μmol/L, respectively） compared to the corresponding chain analogue 4（IC55=24.42 μmol/L）. The results suggest that the aziridine derivatives can get potential activities against nucleophilic methionine synthase.
DENG XilingGUO YingTIAN ChaoLIU JunyiWANG XiaoweiZHANG Zhili
Three series of novel anti-immunodeficiency virus 1 （HIV-1） dual （RT/1N） inhibitors were rationally designed by introducing a functioning diketo acid （DKA） into pyridin-2-one scaffold. To efficiently analyze inhibitory activity, these compounds were screened against HIV-1 RT and IN respectively via surface plasmon resonance （SPR）, and active compounds were subsequently evaluated by enzyme assay. It was noteworthy that compound A2 exhibited moderate activity against both HIV-1 RT and IN. This result provided information for further development of pyridinone analogues as potent dual HIV-1 inhibitors.
With rapid spread of HIV(human immunodeficiency virus) on a global scale and increasingly severe drug-resistance of it,it is urgently necessary to develop novel effective anti-HIV drugs.Non-nucleoside reverse transcriptase inhibitor(NNRTIs)is one of the most significant antiretroviral drugs for fighting against HIV infection due to their various structures,unique mode of action,good efficacy and low toxicity.Pyridinone derivatives,a type of NNRTIs,have been reported to achieve remarkable development in the past few decades.In this review,we summarized current drug design and medicinal chemistry efforts toward the development of next-generation pyridinones as HIV-1 NNRTIs.
