新的核苷类化合物β-L-D4A的化学合成及体外抗HBV作用

目的以D型谷氨酸为原料,通过一系列化学转化,合成了新的核苷类化合物βLD4A,并初步探索其体外抗HBV作用。方法合成βLD4A,用红外光谱、核磁共振氢谱和质谱确证目标化合物的结构,以2.2.15细胞(HepG2细胞进行HBV基因组转染后所得)培养为基础,Southern印迹法检测不同浓度化合物体外抑制HNVDNA复制作用,并求出50%抑制的药物浓度,即EC50。以四噻唑蓝(MTT)比色分析法检测不同浓度药物的细胞毒性,求出IC50。结果化合物βLD4A经红外光谱、核磁共振氢谱和质谱确证;2.2.15细胞培养上清液病毒DNA的Southern印迹、自显影结果显示病毒的抑制呈明显的浓度依赖性,计算出EC50为0.2μmol·L-1,胞内DNA的Southern印迹、自显影显示类似的结果;细胞毒性实验显示IC50为200μmol·L-1。结论体外实验显示βLD4A具有明显的抑制病毒DNA复制作用,且无明显的细胞毒性,TI值为1000,高于临床用Lamivudine(750),有望开发为临床抗HBV用药。

新型核苷类似物β-L-D4A对人DNA聚合酶β和δ的作用及其机制

目的:对人DNA聚合酶δ进行纯化和鉴定,通过检测β-L-D4A对人DNA聚合酶β和δ的作用,观察β-L-D4A的毒副作用.方法:运用多次离子交换层析的方法提取、纯化人Hela细胞的DNA聚合酶δ;检测β-L- D4A对人DNA聚合酶β和δ的酶动力学作用.结果:提纯并鉴定了DNA聚合酶δ,收得率是15%,比活力是1911 Ukat/mg.β-L-D4A为抑制剂时,对人DNA聚合酶β和δ的酶动力学参数分别是Km=1.86 umol/L,2.45 umol/L,IC50 =25.21μmol/L和150.1μmol/L,Ki=24.03μmol/L和132.7μmol/L,结果均提示β-L-D4A对DNA聚合酶β和δ的抑制作用远小于拉米夫定.结论:p-L-D4A是人DNA聚合酶β和δ的竞争性抑制剂,他对这两种酶的毒副作用远小于拉米夫定,可望成为更高效低毒的抗HBV类药物.

新的核苷类似物β-L-D4A的细胞内代谢与生物转化

目的:研究β-L-D4A在2.2.15细胞内的代谢情况,以便为进一步明确其抗HBV作用机制和动物体内实验及人体内试验研究其抗HBV作用与药代动力学奠定基础。方法:以2μmol·L-1[3H]βLD4A或[3H]βDD4A处理2.2.15细胞2、4、8、12、24h,再以0.4mol·L-1预冷的高氯酸(含0.08mol·L-1磷酸三乙胺)抽提,离心后,溶解于酸的上清液,直接进行HPLC分离、相连的紫外检测器检测,然后分析各个峰值。结果:两化合物均出现4峰,且出现时间一致,保留时间依次在6、10、19、28min,24h处理后,β-L-D4A细胞内代谢峰明显高于βDD4A的代谢峰;β-L-D4A于2.2.15细胞内代谢进行了动态的检测,发现细胞内代谢物的含量于加药后迅速增加,以三磷酸形式增加最快,至8h时,达到最大值,以后开始缓慢降低;β-L-D4A处理2.2.15细胞24h后,换用不含药培养基继续培养,停药后3种磷酸化代谢物含量于前8h迅速减少,继后16h降低缓慢,于24h时,仍有8h时35.6%(0.320.9)的三磷酸化合物存在。结论:β-L-D4A较之β-D-D4A更易被磷酸化代谢;一磷酸化过程可能为限速步骤;β-L-D4A三磷酸化物在2.2.15细胞内降解缓慢。

Inhibition of hepatitis B virus by a novel L-nucleoside,β-L-D4A and related analogues

AIM: To explore the inhibition of β-L-D4A on hepatitis B virus (HBV) in 2.2.15 cells derived from HepG2 cells transfected with HBV genome.METHODS: 2.2.15 cells were plated at a density of 5×10^4 per well in 12-well tissue culture plates, and treated with various concentrations of β-L-D4A for 6 days. In the end,5μl of medium was used for the estimation of HBsAg and HBeAg, the other medium was processed to obtain virions by a polyethlene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with HindⅢ. Both DNAs were subjected to Southern blot,hybridized with a ^32P-labeled HBV probe and autoradiophed.Intensity of the autoradiographic bands was quantitated by densitometric scans of computer and ED50 was calculated. Then Hybond-N membrane was washed and rehybridized with a ^32P-labeled mtDNA-specific probe, and effect of β-L-D4A on mitochondrial DNA was studied. 2.2.15 cells were also seeded in 24-well tissue culture plates,and cytotoxicity with different concentrations was examined by MTT method. ID50 was calculated. Structure-activity relationships between D2A and D4A were also studied as above.RESULTS: Autoradiographic bands were similar between supernatant and intracellular HBV DNA. Episomal HBV DNAwas inhibited in a dose-dependent manner. ED50 was 0.2μM. HBsAg or HBeAg was not apparently decreased, and inhibition of mitochondrial DNA was not obvious. The experiment of cytotoxicity gained ID50 at 200 μM.CONCLUSION: β-L-D4A possesses potent inhibitory effects on the replication of HBV in vitro with little cytotoxidty and mitochondrial toxicity, TI value is 1000. It is expected to be developed as a new clinically anti-HBV drug.

Effect and mechanism of β-L-D4A on DNA polymerase α

AIM:To investigate the safety of β-L-D4A on DNA polymeraseα. METHODS:Ion exchange chromatography was used to separate DNA polymeraseαfrom crude extract of human Hela cells.Detailed kinetic parameters were determined for β-L-D4A against DNA polymeraseα. RESULTS:DNA polymeraseαwas purified with 4% yield and 31000 units/rag specific activity.The Michaelis constant (Km=3.22μmol/L),50% inhibition concentration (IC50=178.49μmol/L) and inhibition constant (Ki=126μmol/L) of β-L-D4A were determined by kinetic analysis. CONCLUSION:β-L-D4A is a more safe nucleoside for hepatitis B virus (HBV) infection with a lower host toxicity.

β-L-D4A体外抗乙型肝炎病毒作用及其机制

目的 探索β-L-D4A体外抗乙型肝炎病毒(HBV)作用及其靶点,以明确其抗HBV复制作用的机制。 方法 β-L-D4A干预培养2.2.15细胞后,检测其抗HBV作用;提取细胞内复制核心颗粒,通过内源性聚合酶反应和活性胶实验检测聚合酶和逆转录酶的活性。 结果 β-L-D4A体外明显抑制HBV DNA的复制,并呈现浓度依赖性;内源性聚合酶反应显示β-L-D4A对聚合酶和逆转录酶活性均存在抑制作用,其IC50分别为0.51 μmol/L和0.5 5 μmol/L;外源性核酸为模板的活性胶实验显示聚合酶和逆转录酶活性无变化。 结论 β-L-D4A体外抗HBV作用环节可能在于其代谢物对HBV DNA复制的始动或DNA链延伸过程的不可逆抑制。需行引物结合实验进一步验证。

新型抗HBV化合物:-βL-D4A对DNA聚合酶δ的作用及其机制

目的对DNA聚合酶δ进行纯化和鉴定,通过检测β-L-D4A对DNA聚合酶δ的作用,观察β-L-D4A的毒副作用。方法运用多次离子交换层析的方法提取、纯化人Hela细胞的DNA聚合酶δ;检测β-L-D4A对DNA聚合酶δ的酶动力学作用。结果提纯并鉴定了DNA聚合酶δ,收得率是15%,比活力是1 911 ukat/mg。β-L-D4A为抑制剂时,Km是2.45μmol/L,IC50是150.1μmol/L,K i是132.7μmol/L,均提示β-L-D4A对DNA聚合酶δ的抑制作用远小于拉米夫定。结论β-L-D4A是DNA聚合酶δ的竞争性抑制剂,它对该酶的毒副作用远小于拉米夫定,可望成为更高效低毒的抗HBV类药物。