Structure-based design of novel HCV NS5B thumb pocket 2 allosteric inhibitors with submicromolar gt1 replicon potency: Discovery of a quinazolinone chemotype

We describe the structure-based design of a novel lead chemotype that binds to thumb pocket 2 of HCV NS5B polymerase and inhibits cell-based gt1 subgenomic reporter replicons at sub-micromolar concentrations (EC₅₀ <200 nM). This new class of potent thumb pocket 2 inhibitors features a 1H-quinazolin-4-one scaffold derived from hybridization of a previously reported, low affinity thiazolone chemotype with our recently described anthranilic acid series. Guided by X-ray structural information, a key NS5B–ligand interaction involving the carboxylate group of anthranilic acid based inhibitors was replaced by a neutral two-point hydrogen bonding interaction between the quinazolinone scaffold and the protein backbone. The in vitro ADME and in vivo rat PK profile of representative analogs are also presented and provide areas for future optimization of this new class of HCV polymerase inhibitors.     

Design and synthesis of l- and d-phenylalanine derived rhodanines with novel C5-arylidenes as inhibitors of HCV NS5B polymerase

Hepatitis C virus (HCV) NS5B polymerase is a key target for anti-HCV therapeutics development. Herein, we report the synthesis and in vitro evaluation of anti-NS5B polymerase activity of a molecular hybrid of our previously reported lead compounds 1 (IC₅₀ = 7.7 μM) and 2 (IC₅₀ = 10.6 μM) as represented by hybrid compound 27 (IC₅₀ = 6.7 μM). We have explored the optimal substituents on the terminal phenyl ring of the 3-phenoxybenzylidene moiety in 27, by generating a set of six analogs. This resulted in the identification of compound 34 with an IC₅₀ of 2.6 μM. To probe the role of stereochemistry towards the observed biological activity, we synthesized and evaluated the d-isomers 41 (IC₅₀ = 19.3 μM) and 45 (IC₅₀ = 5.4 μM) as enantiomers of the l-isomers 27 and 34, respectively. The binding site of compounds 32 and 34 was mapped to palm pocket-I (PP-I) of NS5B. The docking models of 34 and 45 within the PP-I of NS5B were investigated to envisage the molecular mechanism of inhibition.     

H5N1重组禽流感病毒样颗粒免疫原性研究

目的对构建的H5N1重组禽流感病毒样颗粒(VLPs)进行初步免疫原性探讨,并与H5N1全病毒灭活疫苗(WIV)进行体液免疫和细胞免疫的比较。方法在0周和3周分别以纯化H5N1重组禽流感病毒样颗粒、H5N1全病毒灭活疫苗及pH7.2 PBS腿部肌肉注射BALB/c小鼠,于不同时间收集血清,以血凝抑制试验(HI)和血清IgG抗体酶联免疫吸附试验(ELISA)评估体液免疫,CD4+、CD8+T细胞亚群及酶联免疫斑点试验(ELISPOT)评估细胞免疫,并以同型毒株滴鼻攻击,观察小鼠存活率。结果病毒样颗粒各组和全病毒灭活疫苗免疫后小鼠血清ELISA IgG效价均有升高;中和抗体效价除病毒样颗粒120 ng/只免疫剂量外其他免疫小鼠HI效价均达1︰40;小鼠脾CD4+T淋巴细胞亚群分类:全病毒灭活疫苗组(600μg/只)为36.56%;病毒样颗粒组(120 ng/只,600 ng/只,2 500 ng/只)分别为26.58%,32.20%,29.25%;PBS组为26.65%;CD8+T淋巴细胞亚群分类:全病毒灭活疫苗组(600 ng/只)为10.78%;病毒样颗粒组(120 ng/只,600 ng/只,2 500 ng/只)分别为1 3.53%,14.24%,1 3.35%;PBS组为10.69%。ELISPOT试验统计学数据显示,病毒样颗粒和全病毒灭活疫苗的小鼠脾单个核细胞分泌IFN-γ细胞与PBS组有显著性差异;小鼠保护性试验结果显示,除病毒样颗粒120 ng/只免疫剂量小鼠的存活率为87.5%外,其他病毒样颗粒实验组小鼠均为100%,PBS对照组为12.5%。结论 H5N1重组禽流感病毒样颗粒能诱导体液免疫和细胞免疫,并能抵御同型病毒株的攻击,可作为H5N1人用禽流感的候选疫苗。     

Cobalt-chelated magnetic particles for one-step purification and immobilization of His6-tagged Escherichia coli γ-glutamyltranspeptidase

Cobalt-chelated magnetic (Fe₃O₄-AA-ANTA-Co^{2 +}) particles were prepared and one-step purification and immobilization of His₆-tagged Escherichia coli γ-glutamyltranspeptidase (His₆-EcGGT) using these particles were evaluated. The optimal conditions for the adsorption of His₆-EcGGT to Fe₃O₄-AA-ANTA-Co^{2 +} particles were found to be 24.7 U g^–1 adsorbent, pH 6.5, 300 mM NaCl and 30 min incubation at 4°C, while the elution solution was optimized to be 50 mM phosphate buffer (pH 6.5) containing 150 mM imidazole and 300 mM NaCl. The immobilized His₆-EcGGT was recycled five times without significant loss of GGT activity. The average yield rate for the synthesis l-theanine from glutamine and ethylamine reached 56.7%. These results indicate that one-step affinity purification and immobilization of His₆-EcGGT by Fe₃O₄-AA-ANTA-Co^{2 +} particles might serve as an effective process for industrial application.     

Preparation of Fine Magnetic Particles and Application for Enzyme Immobilization

Fine magnetic particles (ferrofluid) were prepared from a co-precipitation method by oxidation of Fe²⁺ with nitrite. The particles were activated with (3-aminopropyl)triethoxysilane in toluene and the activated particles were combined with some enzymes by using glutaraldehyde. Enzyme-immobilized magnetic particles were between 4-70 nm and the size could be changed corresponding to the ratio of the amount of Fe²⁺ to that of nitrite. In the immobilization of β-glucosidase, activity yield was 83% and 168 mg protein was immobilized per g magnetite. Other enzymes or proteins could be immobilized at the level between about 70 and 200mg/g support. Immobilized β-glucosidase was stable at 4°C. Magnetic particles immobilized with β-glucosidase responded quickly to the magnetic field and “ON-OFF” control of the enzyme reaction was possible.     

Synthesis and Antiviral Properties of Novel Tetracyclic Nucleoside Inhibitors of Hepatitis C NS5B Polymerase

As part of an ongoing medicinal chemistry effort to identify novel nucleoside inhibitors of HCV NS5B polymerase, we report the discovery of a novel series of 2′-C-Methyl-ribose nucleoside derivatives bearing a 7-aryl and 7-heteroaryl- substituted 7-deaza-adenine nucleobase. A reliable platform for the synthesis and simplified purification of the corresponding nucleoside triphosphates (NTPs) was established, enabling a solid understanding of the SAR relationship within the series. By this approach, we identified the novel analogs 13a and 13b that demonstrated micromolar levels of cellular activity, and the NTPs of which, 16a and 16b, are excellent inhibitors of NS5B with IC₅₀ = 0.1 μM, a level of intrinsic potency similar to that of previous and current clinical candidates.     

复发性妇科恶性肿瘤腹腔镜下病灶切除术^125I粒子植入后血清CA125水平变化及临床意义

目的：研究复发性妇科恶性肿瘤腹腔镜下病灶切除术^125I粒子植入后血清CA125水平变化及临床意义。方法：选取2012年4月-2013年4月来我院就诊的40例复发妇科恶性肿瘤患者,平均分为实验组和对照组,每组各20例。实验组20例行腹腔镜下切除病灶并进行^125I粒子植入治疗,对照组20例仅行腹腔镜切除病灶治疗。监测术后6个月内患者血清CA125的变化情况。结果：术后随访的6个月内,实验组的血清CA125水平明显降低,术后3个月降低至正常水平共有14例（75％）;术后随访的6个月内,对照组血清CA125水平降低后逐步升高,术后3个月降低至正常水平共有3例（15％）,两组比较有统计学意义（P〈0．05）;同时实验组的2a肿瘤生存率为20％,对照组为80％,两组比较有统计学意义（P〈0．05）。结论：复发性妇科恶性肿瘤腹腔镜下病灶切除术125I粒子植入后血清CA125水平显著降低,该法是评估肿瘤治疗疗效的有效方法。     

Interstitial contacts in an RNA-dependent RNA polymerase lattice

Catalytic activities can be facilitated by ordered enzymatic arrays that co-localize and orient enzymes and their substrates. The purified RNA-dependent RNA polymerase from poliovirus self-assembles to form two-dimensional lattices, possibly facilitating the assembly of viral RNA replication complexes on the cytoplasmic face of intracellular membranes. Creation of a two-dimensional lattice requires at least two different molecular contacts between polymerase molecules. One set of polymerase contacts, between the “thumb” domain of one polymerase and the back of the “palm” domain of another, has been previously defined. To identify the second interface needed for lattice formation and to test its function in viral RNA synthesis, we used a hybrid approach of electron microscopic and biochemical evaluation of both wild-type and mutant viral polymerases to evaluate computationally generated models of this second interface. A unique solution satisfied all constraints and predicted a two-dimensional structure formed from antiparallel arrays of polymerase fibers that use contacts from the flexible amino-terminal region of the protein. Enzymes that contained mutations in this newly defined interface did not form lattices and altered the structure of wild-type lattices. When reconstructed into virus, mutations that disrupt lattice assembly exhibited growth defects, synthetic lethality or both, supporting the function of the oligomeric lattice in infected cells. Understanding the structure of polymerase lattices within the multimeric RNA-dependent RNA polymerase complex should facilitate antiviral drug design and provide a precedent for other positive-strand RNA viruses.     

Human immunodeficiency virus-1 Rev protein activates hepatitis C virus gene expression by directly targeting the HCV 5'-untranslated region

Coinfection with human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) accelerates hepatitis C disease progression; however, the mechanism underlying this effect is unknown. Here, we investigated the role of HIV-1 in HCV gene expression and the mechanism involved in this regulation. We discovered that HIV-1 Rev protein activates HCV gene expression. We further revealed that Rev binds to the internal loop of the HCV 5′-untranslated region (5′-UTR) to stimulate HCV IRES-mediated translation.