蛹虫草中抗HIV-1活性成分的研究

以蛹虫草子实体为材料,经水提醇沉后获得沉淀样品 P 和上清样品 L,初步抗 HIV-1蛋白酶检测显示活性成份集中在样品 L 中.进一步利用大孔吸附树脂和高压液相对 L 进行分离纯化,并对纯化样品的体外抗HIV-1蛋白酶活性和抗 HIV-1逆转录酶活性进行了测定.结果显示,L 经大孔吸附树脂和高压液相分离纯化后得到三种物质均具有抗 HIV-1蛋白酶活性 f1,f2和 f3,其中 f1对 HIV-1蛋白酶抑制作用最强,比 L 提高了约29%;三种物质对 HIV-1逆转录酶均有一定的抑制作用.

槐花提取化合物K3体外抗HIV-1活性的研究

目的:研究槐花提取化合物K3的体外抗H IV-1活性,并对其抗H IV-1机制进行初步探讨。方法:采用MTT比色法检测化合物对各种细胞的毒性。用合胞体形成计数法,p24抗原捕获ELISA法及RT-PCR等多种方法研究化合物体外抗H IV-1活性。结论:槐花提取化合物K3体外有较好的抗H IV-1活性,能够抑制病毒实验株(H IV-1ⅢB,耐药株(H IV-174V)和临床分离株(H IV-1KM018)等多种病毒株的复制,且其作用机制是多靶点的,不仅可以抑制病毒的进入,还可以抑制H IV-1逆转录酶活性。

A convenient synthesis of 1-alkyl-5-amino-6-phenylethyluracils as potential non-nucleoside HIV-1RT inhibitors

1-Alkyl-5-amino-6-phenylethyluracils （1a, 1b） were synthesized as potential non-nucleoside HIV-1RT inhibitors. A convenient synthetic procedure was developed for the preparation of 1-alkyl-5-amino or 5-aminosubstituted-6-phenylethyluracils, which were synthesized in three or four steps from 6-methyluracil in good yield. The development of a one-pot reaction that simultaneously removed the benzyl protection group and reduced the nitro group greatly improved the yield of the synthesis. Compounds 1a and 1b are analogs of MKC-442, which is an efficient inhibitor of HIV-1 reverse transcriptase, 1a and 1b were tested for their inhibition of HIV-1RT, and moderate activity was found for 1a.

Targeting Divalent Metal Ions at the Active Site of the HIV-1 RNase H Domain: NMR Studies on the Interactions of Divalent Metal Ions with RNase H and Its Inhibitors

HIV-1 reverse transcriptase (RT) RNase H (HIV-RH) is a key target of anti-AIDS drugs. Metal-chelating compounds are an important class of chemicals in pharmacological drug discovery, especially in relation to HIV-RT and the highly-related HIV-integrase. The correlation between the metal-chelating properties and enzyme activities of the metal chelators is always of high scientific interest, as an understanding of this may accelerate the rational optimization of this class of inhibitors. Our NMR data show that Mg2+ and Ca2+ bind specifically to the active site of the RNase H domain and two Mg2+ ions sequentially bind one molecule of RNase H. We also demonstrate here, using saturated and unsaturated tricyclic N-hydroxypyridones designed to block the active site, that the primary binding sites and affinities of divalent metal ions are correlated with the structures of the chelating motifs. Chemical shift perturbation studies of protein/metal-ion/compound ternary complexes also indicate that divalent metal ions play important roles for the specific interaction of the compounds with the RNase H active site.

Structure-based design and optimization lead to the identification of novel dihydrothiopyrano[3,2-d]pyrimidine derivatives as potent HIV-1 inhibitors against drug-resistant variants

With our continuous endeavors in seeking potent anti-HIV-1 agents,we reported here the discovery,biological characterization,and druggability evaluation of a class of nonnucleoside reverse transcriptase inhibitors.To fully explore the chemical space of the NNRTI-binding pocket,novel series of dihydrothiopyrano[3,2-d]pyrimidines were developed by employing the structure-based design strategy.Most of the derivatives were endowed with prominent antiviral activities against HIV-1 wild-type and resistant strains at nanomolar levels.Among them,compound 23h featuring the aminopiperidine moiety was identified as the most potent inhibitor,with EC50values ranging from 3.43 to 21.4 nmol/L.Especially,for the challenging double-mutants F227L+V106A and K103N+Y181C,23h exhibited 2.3-to 14.5-fold more potent activity than the first-line drugs efavirenz and etravirine.Besides,the resistance profiles of 23h achieved remarkable improvement compared to efavirenz and etravirine.The binding target of 23h was further confirmed to be HIV-1 reverse transcriptase.Molecular modeling studies were also performed to elucidate the biological evaluation results and give guidance for the optimization campaign.Furthermore,no apparent inhibition of the major CYP450 enzymes and hERG channel was observed for 23h.Most importantly,23h was characterized by good pharmacokinetic properties and excellent safety in vivo.Collectively,23h holds great promise as a potential candidate for its effective antiviral efficacy and favorable drug-like profiles.

Synthesis and anti-HIV-1 activity evaluation of N-l-alkyl-5-halogeno-6- alkylamino uracils as novel non-nucleoside HIV-I reverse transcriptase inhibitors

N-l-alkyl-5-halogeno-6-alkylamino uracils, which are novel l-[（2-hydroxyethoxy） methyl]-6-（phenylthio） thymine （HEPT） analogues, were synthesized as the selective and potent non-nucleoside human immunodeficiency virus （HIV）-I reverse transcriptase inhibitors. Some of the compounds showed potent inhibitory activity against HIV-1 reverse transcriptase. For instance, compounds ld, lm and In exhibited potent anti-HIV-1 activity with the ICso values of 13.3, 11.7 and 3.15 μM, respectively, which are comparable to that of nevirapine （IC50 8.38 μM）.

稳定表达Makorin环指蛋白1基因对人宫颈癌Siha细胞的影响

目的研究稳定表达Makorin环指蛋白1(MKRN1)基因对人宫颈癌细胞系Siha细胞的影响。方法利用作者构建的稳定表达MKRN1基因的Siha细胞株,观察MKRN1基因对人端粒酶逆转录酶(hTERT)基因mRNA转录表达水平、端粒酶活性以及其对细胞周期的影响。结果转染Siha细胞后,稳定表达MKRN1基因明显抑制细胞hTERT基因mRNA水平的表达,明显抑制细胞中端粒酶活性,并且G1期细胞明显增加,而S期细胞明显减少。结论稳定表达MKRN1基因可特异性降低宫颈癌细胞中hTERT mRNA水平,抑制癌细胞内端粒酶活性,抑制宫颈癌细胞增殖,并且阻止宫颈癌细胞从G1期进入S期,促进细胞凋亡。

Docking and field-based QSAR studies of S-DABOs as HIV-1 reverse transcriptase inhibitors

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.

Synthesis and anti-HIV-1 activity evaluation of N-1-alkyl-5-halogeno-6-alkylamino uracils as novel non-nucleoside HIV-1 reverse transcriptase inhibitors

N-1-alkyl-5-halogeno-6-alkylamino uracils,which are novel 1-[(2-hydroxyethoxy) methyl]-6-(phenylthio) thymine (HEPT) analogues,were synthesized as the selective and potent non-nucleoside human immunodeficiency virus(HIV)-1 reverse transcriptase inhibitors.Some of the compounds showed potent inhibitory activity against HIV-1 reverse transcriptase.For instance,compounds 1d,1m and 1n exhibited potent anti-HTV-1 activity with the IC_(50) values of 13.3,11.7 and 3.15μM,respectively, which are comparable to that of nevirapine(IC_(50) 8.38μM).

Synthesis and biological evaluation of novel 1-aryl-5-iodo-6-benzyluracils as potent HIV-1 non-nucleoside reverse transcriptase inhibitors

We have synthesized the novel compounds 1a-1i,which are a series of hybrid analogues to 6-benzyl-1-（benzyloxymethyl）- 5-iodouracil,a compound showing strong activity against HIV-1.We also evaluated the activity of these compounds as the inhibitors of HIV-1 reverse transcriptase（HIV-1 RT）,and they have demonstrated moderate activity.

脑铁调素增高诱导膜铁转运蛋白1及其mRNA的表达下调

目的研究脑室注射铁调素(hepcidin,Hep)对膜铁转运蛋白1(ferroportin1,FP1)及其mRNA表达的调节。方法雄性Wistar大鼠16只,随机分为FP1组及对照组,FP1组脑室注射Hep,对照组脑室注射无菌生理盐水,12h后,分别用免疫组化和RT-PCR法测皮质、海马的FP1及其mRNA的表达。结果脑皮质和海马FP1及其mRNA表达较对照组均明显减少(P<0.01)。结论Hep可直接抑制FP1及其mRNA的表达。

Similar pyridinone compounds with different activities of anti-HIV-1 reverse transcriptase

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.

Synthesis and biological evaluation of novel 2-arylalkylthio-5-iodo-6-benzyl S-DABOs as potent non-nucleoside HIV-1 reverse transcriptase inhibitors

A series of novel dihydro-alkylthio-benzyl-oxopyrimidines （S-DABOs） 7a-f have been designed and synthesized with an efficient method. Biological evaluation of their HIV-1 reverse transcriptase inhibitory activities was performed using Nevirapine （NVP） as a reference compound. Among the series, compound 7d shows the highest reverse transcriptase inhibitory activity, which is better than Nevirapine.

The in vitro inhibitory effect of human neutrophil peptide-1 on human immunodeficiency virus type 1

In order to clarify, the mechanism of inhibition of human neutrophil peptide-1 （ HNP-1 ） on hu- man immunodeficiency vires type 1 （HIV-1 ）, CD4^ ＋ cells were used as the target cells for acute infection with HIV-1, and experiments were peffomed separately with the interaction of different concentrations of HNP-1 with free vires particles, un-infected and infected CD4^＋ cells. The activity of reverse transcriptase （RT） in the supematant of cell cultures of different lots of experiments were then assayed accordingly, and the toxicity effect on human lymphocytic cells MT4 was measured by MTT assay. The experimental results showed that pre-incubation of HNP-1 with the concentrated stock of vires could block the binding of vires to target cells with EC50 of 2.49 μg/ml, while pre-treatment of CD4^＋ cells with HNP- 1 prior to inoculation could reduce the ability of cells to bind vires with EC50 of 20.7 μg/ml. In addition, When culturing the infected CD4^＋ cells in the continuous presence of various concentrations of HNP-1 added immediately after infection, HNP-1 exhibited modest inhibitory effect on viral replication with reduced RT activities in comparison with those of the control group （ P 〈 0.05 at 100 μg/ml of the highest concentration） . No cytotoxieity effect of HNP-1 was observed as demonstrated by MTT assay. These results indicate that HNP-1 exerts anti-HIV activity by at least two levels： direct inactivation of vires particles and effect on the ability of target cells to bind with viruses. The evaluation of two parameters, inhibitoty effect and the cytotoxicity renders HNP-1 an available candidate for anti-HIV therapeutic agent.

The Rational Combination Strategy of Immunomodulatory Latency Reversing Agents and Novel Immunotherapy to Achieve HIV-1 Cure

Human immunodeficiency virus(HIV)-1 infection creates a persistent latent reservoir even after antiretroviral therapy,which is the main barrier to HIV cure.One of the most explored strategies is the use of latent reversal agents(LRAs)to activate HIV latent reservoirs,followed by immunotherapy to remove infected cells.Immunomodulatory LRAs have the dual advantage of activating viral latency and promoting immune cell elimination of HIV-infected cells.The emergence of novel immunotherapies has also enhanced the possibility ofHIV clearance.Here we review the activity and potential mechanisms of immunomodulatory agonists and immunotherapies.The possible combinational strategies to achieve HIV functional cure and the problems encountered using this approach are discussed.

北京市2006年新确认HIV-1感染者耐药突变的流行性研究

目的研究北京市2006年新确认HIV-1感染者毒株的耐药突变本底数据。方法随机选取北京市2006年新确认HIV-1感染者抗凝全血标本50份，提取血浆病毒RNA，用逆转录聚合酶链反应扩增HIV-1 pol区基因片段，并进行序列测定及耐药基因型分析。结果成功扩增出34份标本的pol区基因；在1例样本的蛋白酶编码区检测出1个主要耐药突变，7例样本检测出7个次要耐药突变，主要耐药突变为M46L，毒株是CRF01_AE亚型，次要耐药突变有4种，出现的频率分别为A71T（2个）、A71V（3个）、Q58E（1个）、V11IV（1个）。在14例样本逆转录酶编码区检测出一种或多种核苷类和（或）非核苷类逆转录酶抑制剂耐药突变，9例标本检出核苷类逆转录酶抑制剂耐药突变，出现频率分别为：V118I（42．9％）、M184V（7．1％）、A62V（7．1％）、K70T（7．1％）、K65R（7．1％）、K219N（7．1％）、T69d（7．1％）、V75LV（7．1％）、K219R（7．1％）；10例标本检出核苷类逆转录酶抑制剂耐药突变，出现的频率分别为V106I（35．5％）、Y181C（15．4％）、K103KR（7．7％）、K103R（7．7％）、L100LV（7．7％）、V108I（7．7％）、V179D（7．7％）、V179DV（7．7％）。结论北京市2006年新确认HIV-1感染者毒株中已经存在一定比例耐药突变，有必要定期进行耐药性监测研究。

Design, synthesis and activity evaluation of novel pyridinone derivatives as anti-HIV-1 dual(RT/IN) inhibitors

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.

Pyridin-2(1H)-ones as HIV-1 NNRTIs:a combinatorial optimization strategy

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.

Synthesis and anti-HIV activities of phorbol derivatives

In this study,37 derivatives of phorbol esters were synthesized and their anti-HIV-1 activities evaluated,building upon our previous synthesis of 51 phorbol derivatives.12-Para-electron-acceptor-trans-cinnamoyl-13-decanoyl phorbol derivatives stood out,demonstrating remarkable anti-HIV-1 activities and inhibitory effects on syncytia formation.These derivatives exhibited a higher safety index compared with the positive control drug.Among them,12-(trans-4-fluorocinnamoyl)-13-decanoyl phorbol,designated as compound 3c,exhibited the most potent anti-HIV-1 activity(EC_(50)2.9 nmol·L^(−1),CC50/EC_(50)11117.24)and significantly inhibited the formation of syncytium(EC_(50)7.0 nmol·L^(−1),CC50/EC_(50)4891.43).Moreover,compound 3c is hypothesized to act both as an HIV-1 entry inhibitor and as an HIV-1 reverse transcriptase inhibitor.Isothermal titration calorimetry and molecular docking studies indicated that compound 3c may also function as a natural activator of protein kinase C(PKC).Therefore,compound 3c emerges as a potential candidate for developing new anti-HIV drugs.

The capsid revolution

Lenacapavir,targeting the human immunodeficiency virus type-1(HIV-1)capsid,is the first-in-class antiretroviral drug recently approved for clinical use.The development of Lenacapavir is attributed to the remarkable progress in our understanding of the capsid protein made during the last few years.Considered little more than a component of the virus shell to be shed early during infection,the capsid has been found to be a key player in the HIV-1 life cycle by interacting with multiple host factors,entering the nucleus,and directing integration.Here,we describe the key advances that led to this‘capsid revolution’.