新型非肽类小分子Caspase-3抑制剂的设计、合成、活性检测与分子对接研究
|
摘要
Caspase家族由高度同源的半胱氨酸蛋白酶组成,这些半胱氨酸蛋白酶在介导炎症和细胞凋亡中扮演着重要角色.其中,caspase-3被认为是最重要的凋亡执行者。许多危害人类健康的功能紊乱疾病的病因都牵涉到依赖cmpase-3激活的细胞异常凋亡。因此,cmpase-3被认为是一种有应用价值的药物开发靶标,并且人们也一直在努力的寻找它的高效抑制剂。
由于肽类抑制剂普遍存在着细胞透膜性差,体内代谢稳定性差等问题,所以我们把更多的目光投向了非肽类caspase-3抑制剂。为了寻找具有新结构的小分子非肽类抑制剂,我们前期的主要工作集中于对我们拥有的化合物库进行筛选。丹参酮ⅡA,丹参中主要活性成份中的一种,首先被筛选出来,其半数抑制率(IC_(50))为4.1μM。丹参酮ⅡA磺酸钠,丹参酮ⅡA的水溶性衍生物,也是目前国内的一种上市药品,被检测出有更强的caspase-3抑制活性(IC_(50)=0.36μM)。另外,玫红酸被作为一种全新的caspase-3抑制剂也被筛选出来,其半数抑制率(IC_(50))为0.27μM。
为了得到更有效的caspase-3抑制剂,丹参酮ⅡA磺酸钠和玫红酸被确定为用于结构修饰和模拟的先导化合物。通过对丹参酮ⅡA磺酸钠的结构优化,我们设计并合成出了两种新型的、非肽类小分子caspase-3抑制剂,磺基呋喃酮和硝基呋喃酮。另外通过对玫红酸结构的模拟,我们合成了一种玫红酸的类似物,酚酞甲酯。在体外酶活性检测中,这三种抑制剂都显示出了较高的抑制活性(IC_(50)=0.11μM,0.21μM和0.14μM)。通过对它们分子对接模型的研究,我们对其产生抑制作用的结构基础有了初步了解。此外,在细胞学的初步研究中,硝基呋喃酮在Jurkat T细胞模型中,表现出了较好的细胞保护作用。
综上,在本论文中我们设计合成了两类新型的小分子非肽类抑制剂,它们的发现为将来caspase-3抑制剂的开发提供了一些信息。我们下一步的工作一方面将对现有的抑制剂展开更为深入细致的研究,另外一方面将继续改造现有抑制剂的结构以期进一步提高它们的抑制活力。
The caspase family comprises highly homologous cysteine proteases that play key roles in inflammation and apoptosis.Two different classes of caspases are involved in apoptosis,the initiator caspases and the executioner caspases.The initiator caspases, which include caspase-2,-8,-9,and -10,are located at the top of the signaling cascade; their primary function is to activate the executioner caspases,such as caspase-3,-6, and -7.Among executioner caspases,caspase-3 is activated in caspase-related model of apoptosis and is believed to be the "central executioner" of the apoptotic pathway.
Activation of caspase-3-dependent apoptotic cell death has been implicated in the etiology of many harmful human disorders,such as myocardial infarction, Alzheimer's,stroke,Parkinson's,sepsis,and Huntington's disease.Researchers have also revealed in their studies that caspase-3 inhibitors block apoptotic cell death and improve neurologic outcome.Hence,caspase-3 is believed to be a valuable drug discovery target and great efforts have been made to search for potent inhibitors.
To date,most inhibitors of caspase-3 have been peptidebased compounds that inhibit the catalytic activity of this enzyme.However,one of the potential problems of peptide-based caspase inhibitors is their poor metabolic stability and poor cell penetration,which has resulted in a search for nonpeptide-based inhibitors of caspase-3.
To find out small-molecular nonpeptide caspase-3 inhibitors with novel structures directed against diseases involving abnormally upregulated caspase-3-dependent apoptosis,our work was initially based on screening of a small-molecule library of compounds.TanshinoneⅡA(IC_(50)= 4.1μM),one of the primary effective components from Danshen,was proved to have inhibitory activity to caspase-3 in vitro.A water-soluble derivative of tanshinoneⅡA,sodium tanshinoneⅡA sulfonate (STS) which was a comercially available Chinese medicine,showed much higher inhibitory activity(IC_(50)= 0.36μM) than TanshinoneⅡA.Moreover,4-[bis(4-hydroxyphenyl)methylene]-2,5-cyclohexadien-1-one(rosolic acid) with an IC_(50) of 0.27μM,was identified as a novel inhibitor of caspase-3.This paper was divided into two parts.
PartⅠ
To get more potent caspase-3 inhibitor,STS was used as a leading compound for structural modification.In the present paper,we designed and synthesized 1-Sulfo-6,6-dimethyl-6,7-dihydrobenzofuran-4(5H)-one(SF) and 1-Nitro-6,6-dimethyl-6,7-dihydrobenzofuran-4(5H)-one(NF) as novel small-molecular inhibitors of caspase-3 through structural optimization of STS.The structural optimization depended on computer-assisted drug design.SF and NF showed high inhibitory potency against caspase-3 in vitro(IC_(50)=0.11μM and 0.21μM).This section was divided into six parts:design,synthesis,enzyme assay, molecular docking study,assays for cytotoxicity in Jurkat T cells and assays for antiapoptotic action in Jurkat T cells.
1.Design
In order to design scaffold of STS mimic small molecules,we first analyzed binding mode of STS through a computational docking simulation.STS was located within the active site of caspase-3,and forms eight hydrogen bonds with Arg64, Ser120,His121,Trp204,Ser205,and Arg207.One ketone group in STS adjoined the thiol of Cys163,but hexahydronaphthalin subunit in STS wasn't bound to the S2 subsite(Tyr204,Trp206,and Phe256).The results of binding mode revealed that only one ketone group in furano-o-quinone was necessary to interact with the thiol of Cys163,and hexahydronaphthalin subunit have no hydrophobic interactions with the S2 subsite.
Taking into account these considerations,we first designed a small-molecular analogue of STS,SF,in which hexahydronaphthalin subunit was removed, furano-o-quinone was converted to furano-ketone.Moreover,as a rule,highly ionized sulfosalts cannot cross the biological membranes which are permeable only to non-dissociated molecules.Therefore,sulfonic group was replaced with nitro group and NF was designed.Molecular docking was carried out using the software AUTODOCK4 with low-energy conformer of the ligand obtained with the ChemBio3D ultra 11.0 software[Cambridge Soft Corporation,USA(2007)].During each docking experiment which was carried out with a rigid enzyme receptor,10 runs were carried out.Protein structure was obtained from the Protein Data Bank(1GWF).
2.Synthesis
The designed SF was prepared as follows:(1) Dimedon with chloroacetaldehyde gave a furan derivative.(2) Reaction of the furan derivative with concentrated sulfuric acid in acetic anhydride afforded the final compound SF.
The designed NF was prepared as follows:(1) Dimedon with chloroacetaldehyde gave a furan derivative.(2) Reaction of the furan derivative with fuming nitric acid in acetic anhydride afforded the final compound NF.
3.Enzyme Assay
The synthesized SF and NF have been tested for their ability to inhibit caspase-3 proteolytic activity to breakdown its fluorogenic substrate,DEVD-AFC and displayed high activity in vitro caspase-3 inhibition assay(IC_(50)=0.11μM and 0.21μM).Parallel experiments demonstrated that the IC_(50) value for 5-Nitroisatin,a known nonpeptide inhibitor of caspase-3,was equal to 2.8μM under the same experimental condition. Compared with original STS,SF and NF showed higher inhibitory activity, indicating that the ability of interaction with the active site of caspase-3 has been improved through optimizing the structure of STS.The result also suggested that the inhibitory potency hasn't been diminished after removing one ketone group and hexahydronaphthalin subunit,replacing sulfonic group with nitro group.
4.Molecular docking Study
Molecular docking was carried out using the software AUTODOCK4.Protein structure was obtained from the Protein Data Bank(1GWF).From the docking simulation,NF was located within the active site of caspase-3,and formed ten hydrogen bonds with Arg64,Ser120,Gln161,Ser205,and Arg207;SF was also located within the active site of caspase-3,and formed ten hydrogen bonds with Arg64,Ser120,Gln161,Ser205,and Arg207.These interactions closely resemble those of STS.Additionally,two methyls in SF and NF are bound to the S2 subsite based on hydrophobic interactions,which was inexistence in STS.The docking results of SF and NF with caspase-3 indicated that the designed inhibitors were likely to process good inhibitory potency.
5.Assays for cytotoxicity in Jurkat T Cells.
The cytotoxic effects on Jurkat T cells was evaluated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]assay.NF showed little cytotoxic effect in MTT assay.It was not shown that the cell viability goes down in a NF concentration-dependent manner.Jurkat T cells did not display any significant inhibition of the cell viabilities even incubated with the highest dose of NF(100μM). The results of assay revealed that NF is not toxic to the cells.
6.Assays for antiapoptotic action in Jurkat T Cells.
The antiapoptotic effect of NF was evaluated in camptothecin-treated human Jurkat T cells which is a cell-based model of apoptosis.NF demonstrated significant protection against apoptosis.The ability of NF to inhibit apoptosis was first assessed by staining the cells with DAPI(4',6-diamidino-2-phenylindole) for morphological analysis under fluorescence microscopy.NF inhibited the formation of apoptotic bodies in Jurkat T cells in a concentration-dependent manner.To confirm the antiapoptotic effect of NF another experiment of measuring cell viability with MTT assay was performe.NF improved the cell viability of the apoptotic cells in a concentration dependent manner.These results indicated that NF had a level of protection against apoptosis in the human Jurket T cells.These results also suggested, though indirectly,good cell permeability of NF.In all anti-apoptosis assays, Z-VAD-FMK was used as a positive control in parallel with NF.
PartⅡ
In order to verify the universality of the new structure of rosolic acid,we synthesized the analog of rosolic acid,4-[(4-hydroxyphenyl-2-methoxycarbonylphenyl) methylene]-2,5-cyclohexadien-1-one(phenolphthalein methyl ester). Phenolphthalein methyl ester had similar structure with rosolic acid,and they both belonged to fuchsone derivatives.Fortunately,compared with rosolic acid, phenolphthalein methyl ester showed slightly higher inhibitory potency against caspase-3 in vitro enzyme assay(IC_(50)= 0.14μM).In order to improve our understanding of the interaction between this class of compounds and caspase-3,we also analyzed the binding model and cell model of phenolphthalein methyl ester which showed higher inhibitory potency in vitro caspase-3 assay through a computational docking simulation.This section was divided into five parts:design, synthesis,enzyme assay,molecular docking study and assays for cytotoxicity in Jurkat T cells.
1.Design
In order to design scaffold of rosolic acid mimic small molecules,we first analyzed binding model of rosolic acid through a computational docking simulation. But,from the docking model,we did not get much useful information.Therefore,the rosolic acid mimic small molecules,phenolphthalein methyl ester,was designed by mainly basing on the structural information of rosolic acid.
1.Synthesis
Phenolphthalein methyl ester Was prepared as follows:phenolphthalein reacted with methanol in the presence of thionyl chloride at 55℃for 12h to give phenolphthalein methyl ester in 34%yield.
2.Enzyme Assay
Rosolic acid and phenolphthalein methyl ester had been tested for its ability to inhibit caspase-3 catalyzed proteolytic breakdown of its fluorogenic substrate, DEVD-AFC.Rosolic acid and phenolphthalein methyl ester showed similar inhibitory activity against caspase-3 in vitro(IC_(50)= 0.27μM and 0.14μM).Parallel experiments demonstrated that the IC_(50) value for 5-Nitroisatin,a known nonpeptide inhibitor of caspase-3,was equal to 2.8μM under the same experimental condition. The results indicated that the two fuchsone derivatives demonstrated some level of interaction with the active site of caspase-3.The result also suggested that the two fuchsone derivatives were likely to be a novel caspase-3 inhibitor with possible pharmaceutical application potential.
3.Molecular docking Study
Molecular docking was carried out using the software AUTODOCK4.Protein structure was obtained from the Protein Data Bank(1GWF).From the results of docking simulation,we could see that phenolphthalein methyl ester was located within the active site of caspase-3 and formed two hydrogen bonds with Ser209, which existed only in caspase-3 among all human caspases.The ketone group in phenolphthalein methyl ester was adjacent to the thiol of Cys163.Additionally,one of the three benzene rings in phenolphthalein methyl ester was bound to the S2 subsite (Tyr204,Trp206,and Phe256) based on hydrophobic interactions.
The results of molecular docking suggested phenolphthalein methyl ester actually interacted with the active site;this might be the principal mechanism of inhibition. Moreover,the binding of phenolphthalein methyl ester to caspase-3 provided a good structural basis to explain the efficient inhibitory action of phenolphthalein methyl ester.But,from the results of docking simulation,we also found that the binding models of rosolic acid and phenolphthalein methyl ester,were not exactly the same. The results indicated that their structural basis of inhibition remains to be further studied.
4.Assays for cytotoxicity in Jurkat T Cells.
The cytotoxic effects on Jurkat T cells was also evaluated by the MTT assay.It was shown that the cell viability goes down in a phenolphthalein methyl ester Concentration-dependent manner,indicating that phenolphthalein methyl ester is cytotoxic.The results revealed that the structure of phenolphthalein methyl ester have some shortcomings.The structure of phenolphthalein methyl ester required further refinement.
In summary,sodium tanshinoneⅡA sulfonate(STS),an existing drug,was identified as a lead compound of caspase-3 inhibitor.Novel and potent caspase-3 inhibitors,SF and NF,were discovered through structural simplification of the lead compound.NF had a protection against apoptosis.Rosolic acid and phenolphthalein methyl ester,which belonged to fuchsone derivatives,were identified as novel and potent caspase-3 inhibitors.These two compounds may represent a new class of caspase-3 inhibitors.All of these small-molecule caspase-3 inhibitor with novel structure might provide some information for discovery of anti-caspase-3 inhibitors. Further work was in progress to further modify the structures of these new inhibitors to improve their effectiveness,and to study their anti-caspase-3 activity in vivo.
由于肽类抑制剂普遍存在着细胞透膜性差,体内代谢稳定性差等问题,所以我们把更多的目光投向了非肽类caspase-3抑制剂。为了寻找具有新结构的小分子非肽类抑制剂,我们前期的主要工作集中于对我们拥有的化合物库进行筛选。丹参酮ⅡA,丹参中主要活性成份中的一种,首先被筛选出来,其半数抑制率(IC_(50))为4.1μM。丹参酮ⅡA磺酸钠,丹参酮ⅡA的水溶性衍生物,也是目前国内的一种上市药品,被检测出有更强的caspase-3抑制活性(IC_(50)=0.36μM)。另外,玫红酸被作为一种全新的caspase-3抑制剂也被筛选出来,其半数抑制率(IC_(50))为0.27μM。
为了得到更有效的caspase-3抑制剂,丹参酮ⅡA磺酸钠和玫红酸被确定为用于结构修饰和模拟的先导化合物。通过对丹参酮ⅡA磺酸钠的结构优化,我们设计并合成出了两种新型的、非肽类小分子caspase-3抑制剂,磺基呋喃酮和硝基呋喃酮。另外通过对玫红酸结构的模拟,我们合成了一种玫红酸的类似物,酚酞甲酯。在体外酶活性检测中,这三种抑制剂都显示出了较高的抑制活性(IC_(50)=0.11μM,0.21μM和0.14μM)。通过对它们分子对接模型的研究,我们对其产生抑制作用的结构基础有了初步了解。此外,在细胞学的初步研究中,硝基呋喃酮在Jurkat T细胞模型中,表现出了较好的细胞保护作用。
综上,在本论文中我们设计合成了两类新型的小分子非肽类抑制剂,它们的发现为将来caspase-3抑制剂的开发提供了一些信息。我们下一步的工作一方面将对现有的抑制剂展开更为深入细致的研究,另外一方面将继续改造现有抑制剂的结构以期进一步提高它们的抑制活力。
The caspase family comprises highly homologous cysteine proteases that play key roles in inflammation and apoptosis.Two different classes of caspases are involved in apoptosis,the initiator caspases and the executioner caspases.The initiator caspases, which include caspase-2,-8,-9,and -10,are located at the top of the signaling cascade; their primary function is to activate the executioner caspases,such as caspase-3,-6, and -7.Among executioner caspases,caspase-3 is activated in caspase-related model of apoptosis and is believed to be the "central executioner" of the apoptotic pathway.
Activation of caspase-3-dependent apoptotic cell death has been implicated in the etiology of many harmful human disorders,such as myocardial infarction, Alzheimer's,stroke,Parkinson's,sepsis,and Huntington's disease.Researchers have also revealed in their studies that caspase-3 inhibitors block apoptotic cell death and improve neurologic outcome.Hence,caspase-3 is believed to be a valuable drug discovery target and great efforts have been made to search for potent inhibitors.
To date,most inhibitors of caspase-3 have been peptidebased compounds that inhibit the catalytic activity of this enzyme.However,one of the potential problems of peptide-based caspase inhibitors is their poor metabolic stability and poor cell penetration,which has resulted in a search for nonpeptide-based inhibitors of caspase-3.
To find out small-molecular nonpeptide caspase-3 inhibitors with novel structures directed against diseases involving abnormally upregulated caspase-3-dependent apoptosis,our work was initially based on screening of a small-molecule library of compounds.TanshinoneⅡA(IC_(50)= 4.1μM),one of the primary effective components from Danshen,was proved to have inhibitory activity to caspase-3 in vitro.A water-soluble derivative of tanshinoneⅡA,sodium tanshinoneⅡA sulfonate (STS) which was a comercially available Chinese medicine,showed much higher inhibitory activity(IC_(50)= 0.36μM) than TanshinoneⅡA.Moreover,4-[bis(4-hydroxyphenyl)methylene]-2,5-cyclohexadien-1-one(rosolic acid) with an IC_(50) of 0.27μM,was identified as a novel inhibitor of caspase-3.This paper was divided into two parts.
PartⅠ
To get more potent caspase-3 inhibitor,STS was used as a leading compound for structural modification.In the present paper,we designed and synthesized 1-Sulfo-6,6-dimethyl-6,7-dihydrobenzofuran-4(5H)-one(SF) and 1-Nitro-6,6-dimethyl-6,7-dihydrobenzofuran-4(5H)-one(NF) as novel small-molecular inhibitors of caspase-3 through structural optimization of STS.The structural optimization depended on computer-assisted drug design.SF and NF showed high inhibitory potency against caspase-3 in vitro(IC_(50)=0.11μM and 0.21μM).This section was divided into six parts:design,synthesis,enzyme assay, molecular docking study,assays for cytotoxicity in Jurkat T cells and assays for antiapoptotic action in Jurkat T cells.
1.Design
In order to design scaffold of STS mimic small molecules,we first analyzed binding mode of STS through a computational docking simulation.STS was located within the active site of caspase-3,and forms eight hydrogen bonds with Arg64, Ser120,His121,Trp204,Ser205,and Arg207.One ketone group in STS adjoined the thiol of Cys163,but hexahydronaphthalin subunit in STS wasn't bound to the S2 subsite(Tyr204,Trp206,and Phe256).The results of binding mode revealed that only one ketone group in furano-o-quinone was necessary to interact with the thiol of Cys163,and hexahydronaphthalin subunit have no hydrophobic interactions with the S2 subsite.
Taking into account these considerations,we first designed a small-molecular analogue of STS,SF,in which hexahydronaphthalin subunit was removed, furano-o-quinone was converted to furano-ketone.Moreover,as a rule,highly ionized sulfosalts cannot cross the biological membranes which are permeable only to non-dissociated molecules.Therefore,sulfonic group was replaced with nitro group and NF was designed.Molecular docking was carried out using the software AUTODOCK4 with low-energy conformer of the ligand obtained with the ChemBio3D ultra 11.0 software[Cambridge Soft Corporation,USA(2007)].During each docking experiment which was carried out with a rigid enzyme receptor,10 runs were carried out.Protein structure was obtained from the Protein Data Bank(1GWF).
2.Synthesis
The designed SF was prepared as follows:(1) Dimedon with chloroacetaldehyde gave a furan derivative.(2) Reaction of the furan derivative with concentrated sulfuric acid in acetic anhydride afforded the final compound SF.
The designed NF was prepared as follows:(1) Dimedon with chloroacetaldehyde gave a furan derivative.(2) Reaction of the furan derivative with fuming nitric acid in acetic anhydride afforded the final compound NF.
3.Enzyme Assay
The synthesized SF and NF have been tested for their ability to inhibit caspase-3 proteolytic activity to breakdown its fluorogenic substrate,DEVD-AFC and displayed high activity in vitro caspase-3 inhibition assay(IC_(50)=0.11μM and 0.21μM).Parallel experiments demonstrated that the IC_(50) value for 5-Nitroisatin,a known nonpeptide inhibitor of caspase-3,was equal to 2.8μM under the same experimental condition. Compared with original STS,SF and NF showed higher inhibitory activity, indicating that the ability of interaction with the active site of caspase-3 has been improved through optimizing the structure of STS.The result also suggested that the inhibitory potency hasn't been diminished after removing one ketone group and hexahydronaphthalin subunit,replacing sulfonic group with nitro group.
4.Molecular docking Study
Molecular docking was carried out using the software AUTODOCK4.Protein structure was obtained from the Protein Data Bank(1GWF).From the docking simulation,NF was located within the active site of caspase-3,and formed ten hydrogen bonds with Arg64,Ser120,Gln161,Ser205,and Arg207;SF was also located within the active site of caspase-3,and formed ten hydrogen bonds with Arg64,Ser120,Gln161,Ser205,and Arg207.These interactions closely resemble those of STS.Additionally,two methyls in SF and NF are bound to the S2 subsite based on hydrophobic interactions,which was inexistence in STS.The docking results of SF and NF with caspase-3 indicated that the designed inhibitors were likely to process good inhibitory potency.
5.Assays for cytotoxicity in Jurkat T Cells.
The cytotoxic effects on Jurkat T cells was evaluated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]assay.NF showed little cytotoxic effect in MTT assay.It was not shown that the cell viability goes down in a NF concentration-dependent manner.Jurkat T cells did not display any significant inhibition of the cell viabilities even incubated with the highest dose of NF(100μM). The results of assay revealed that NF is not toxic to the cells.
6.Assays for antiapoptotic action in Jurkat T Cells.
The antiapoptotic effect of NF was evaluated in camptothecin-treated human Jurkat T cells which is a cell-based model of apoptosis.NF demonstrated significant protection against apoptosis.The ability of NF to inhibit apoptosis was first assessed by staining the cells with DAPI(4',6-diamidino-2-phenylindole) for morphological analysis under fluorescence microscopy.NF inhibited the formation of apoptotic bodies in Jurkat T cells in a concentration-dependent manner.To confirm the antiapoptotic effect of NF another experiment of measuring cell viability with MTT assay was performe.NF improved the cell viability of the apoptotic cells in a concentration dependent manner.These results indicated that NF had a level of protection against apoptosis in the human Jurket T cells.These results also suggested, though indirectly,good cell permeability of NF.In all anti-apoptosis assays, Z-VAD-FMK was used as a positive control in parallel with NF.
PartⅡ
In order to verify the universality of the new structure of rosolic acid,we synthesized the analog of rosolic acid,4-[(4-hydroxyphenyl-2-methoxycarbonylphenyl) methylene]-2,5-cyclohexadien-1-one(phenolphthalein methyl ester). Phenolphthalein methyl ester had similar structure with rosolic acid,and they both belonged to fuchsone derivatives.Fortunately,compared with rosolic acid, phenolphthalein methyl ester showed slightly higher inhibitory potency against caspase-3 in vitro enzyme assay(IC_(50)= 0.14μM).In order to improve our understanding of the interaction between this class of compounds and caspase-3,we also analyzed the binding model and cell model of phenolphthalein methyl ester which showed higher inhibitory potency in vitro caspase-3 assay through a computational docking simulation.This section was divided into five parts:design, synthesis,enzyme assay,molecular docking study and assays for cytotoxicity in Jurkat T cells.
1.Design
In order to design scaffold of rosolic acid mimic small molecules,we first analyzed binding model of rosolic acid through a computational docking simulation. But,from the docking model,we did not get much useful information.Therefore,the rosolic acid mimic small molecules,phenolphthalein methyl ester,was designed by mainly basing on the structural information of rosolic acid.
1.Synthesis
Phenolphthalein methyl ester Was prepared as follows:phenolphthalein reacted with methanol in the presence of thionyl chloride at 55℃for 12h to give phenolphthalein methyl ester in 34%yield.
2.Enzyme Assay
Rosolic acid and phenolphthalein methyl ester had been tested for its ability to inhibit caspase-3 catalyzed proteolytic breakdown of its fluorogenic substrate, DEVD-AFC.Rosolic acid and phenolphthalein methyl ester showed similar inhibitory activity against caspase-3 in vitro(IC_(50)= 0.27μM and 0.14μM).Parallel experiments demonstrated that the IC_(50) value for 5-Nitroisatin,a known nonpeptide inhibitor of caspase-3,was equal to 2.8μM under the same experimental condition. The results indicated that the two fuchsone derivatives demonstrated some level of interaction with the active site of caspase-3.The result also suggested that the two fuchsone derivatives were likely to be a novel caspase-3 inhibitor with possible pharmaceutical application potential.
3.Molecular docking Study
Molecular docking was carried out using the software AUTODOCK4.Protein structure was obtained from the Protein Data Bank(1GWF).From the results of docking simulation,we could see that phenolphthalein methyl ester was located within the active site of caspase-3 and formed two hydrogen bonds with Ser209, which existed only in caspase-3 among all human caspases.The ketone group in phenolphthalein methyl ester was adjacent to the thiol of Cys163.Additionally,one of the three benzene rings in phenolphthalein methyl ester was bound to the S2 subsite (Tyr204,Trp206,and Phe256) based on hydrophobic interactions.
The results of molecular docking suggested phenolphthalein methyl ester actually interacted with the active site;this might be the principal mechanism of inhibition. Moreover,the binding of phenolphthalein methyl ester to caspase-3 provided a good structural basis to explain the efficient inhibitory action of phenolphthalein methyl ester.But,from the results of docking simulation,we also found that the binding models of rosolic acid and phenolphthalein methyl ester,were not exactly the same. The results indicated that their structural basis of inhibition remains to be further studied.
4.Assays for cytotoxicity in Jurkat T Cells.
The cytotoxic effects on Jurkat T cells was also evaluated by the MTT assay.It was shown that the cell viability goes down in a phenolphthalein methyl ester Concentration-dependent manner,indicating that phenolphthalein methyl ester is cytotoxic.The results revealed that the structure of phenolphthalein methyl ester have some shortcomings.The structure of phenolphthalein methyl ester required further refinement.
In summary,sodium tanshinoneⅡA sulfonate(STS),an existing drug,was identified as a lead compound of caspase-3 inhibitor.Novel and potent caspase-3 inhibitors,SF and NF,were discovered through structural simplification of the lead compound.NF had a protection against apoptosis.Rosolic acid and phenolphthalein methyl ester,which belonged to fuchsone derivatives,were identified as novel and potent caspase-3 inhibitors.These two compounds may represent a new class of caspase-3 inhibitors.All of these small-molecule caspase-3 inhibitor with novel structure might provide some information for discovery of anti-caspase-3 inhibitors. Further work was in progress to further modify the structures of these new inhibitors to improve their effectiveness,and to study their anti-caspase-3 activity in vivo.
引文
[1]O'Brien T.Prospects for caspase inhibitors[J].Mini ReV.Med.Chem,2004,4,153.
[2]Osman A,Ismail B,Alper K,et al.Therapeutic efficacy of Ac-DMQD-CHO,a caspase 3 inhibitor,for rat spinal cord injury[J].J.Clin.Neurosci,2008,15,672.
[3]Junichi S,Atsushi Y,Yasuyo N,et al.Structure-based discovery of a novel non-peptidic small molecular inhibitor of caspase-3[J].Bioorg.Med.Chem.,2008,16,4854.
[4]Zhang Y,Center D M,Wu D M,et al.processing and activation of the pro-interleukin-1β by caspase-3[J].J Biol Chem,1998,273(2):1144-1148.
[5]熊世勤,来锡华.Caspase激活与调控的分子机制[J].生物化学与生物物理进展,2002,27(10):33-36。
[6]王筱冰,张小翠,夏妙红,等.Caspase的活化机制[J].现代生物医学进展,2006,6(3):53-55。
[7]Chang HY et al.Proteases for Cell Suicide:Function and Regulation of Caspase.Microbiol[J].Mol.Biol.Rev,2000,64:821-846.
[8]Thornberry N.A,Rano T.A,Peterson E.P,et al.,A combinatorial approach defines specifieities of members of the caspase family and granzyme B.Functional relationships established for key mediators of apoptosis[J].J.Biol.Chem,1997,272,17907.
[9]Carcia-Calvo M,Peterson E.P,Rasper D.M,et al.Purification and catalytic properties of human caspase family members[J].Cell Death Differ,1999,6,362.
[10]Pistritto G.,Jost M,Srinivasula S.M,et al.Expression and transcriptional regulation of easpase-14 in simple and complex epithelia[J].Cell Death Differ,2002,9,995.
[11]Denault J.B,Salvesen G.S,Caspases:keys in the ignition of cell death[J].Chem.Rev,2002,102,4489
[12]O'Brien T,Lee D.Prospects for caspase inhibitors[J].Mini Rev.Med.Chem,2004,4,153.
[13]Meier P,Finch A,Evan G,Apoptosis in development[J].Nature,2000,407,796.
[14]Armstrong R.C,Aia T.J,Hoang K.D,et al.Activation of the CED3/ICE-related protease CPP32 in cerebellar granule neurons undergoing apoptosis but not necrosis[J].J.Neurosci.,1997,17,553.
[15]Yuan J,Shaham J,Ledoux S,et al.The C.elegans cell death gene ted-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme[J].Cell,1993,75,641.
[16]易成蜡,陈安民.脊柱损伤的细胞凋亡及其调控.中华创伤杂质[J].2003;19(3):190-192.
[17]刘旭,徐平.心肌缺血-再灌注损伤与细胞凋亡[J].中华实用中西医杂志.2005,18(18):981-983.
[18]Saraste A,et al.Apoptosis in human acute myocardial infarction.Circulation[J],1997,95:320-323.
[19]马永刚,刘世清,彭昊,等.硫酸镁对实验性脊髓损伤的保护作用[J].医学研究生学报,2002;15(6):490-492.
[20]Yan Zhang,Cynthia Goodyer,and Andren Le Blanc.Selective andprotracted apoptosis in human primary neurons microinjection withactive caspase-3,-6,-7and -8[J].J Neurosci,2000,20:8384-8389
[21]Cryns V,Yuan J.Protease to die for[J].Genes Dev,1998,12(11):1551-1570.
[22]Communal C,Sumandea M,De Tombe P,et al.Functional consequences of caspase activation in cardiac myocytes[J].Proc Natl Acad Sci USA,2002,99(9):6252-6256.
[23]Enari M,Sakahira H,Yokoyama H,et al.A caspase-activated DNase that degrades DNA during apoptosis,and its inhibitor ICAD[J].Nature,1998,391:43.
[24]Xiao F,Widlak P,Garrard WT.Engineered apoptotic nucleases for chromatin research[J].Nucleic Acids Res,2007,35(13):e93.
[25]Basnakian AG,Ueda N,Hong X,et al.Ceramide synthase is essential for endonuclease-mediated death of renal tubular epithelial cells induced by hypoxia-reoxygenation[J].Am J Physiol Renal Physiol,2005,288(2):F308-314.
[26]Ye H,Cande C,Stephanou NC,et al.DNA binding is required for apoptogenic action of apoptosis inducing factor[J].Nat Struct Biol,2002,9:680-684.
[27]王筱冰,张小翠,夏妙红,等.Caspase的活化机制[J].现代生物医学进展,2006,6(3):53-55.
[28]Kukhta VK,Marozldna NV,Sokolchik IG,et al.Molecular mechanisms of apoptosis[J].Ukr Biokhim Zh,2003,75(6):5-9.
[29]Diep QN,EI Mabrouk M,Yue P,et al.Effect of AT(1) receptor blockade on cardiac apoptosis in angiotensin-induced hypertension[J].Am J Physiol Heart Circ Physiol,2002,282(5):H1635-1641.
[30]Endres M,Namura S,Shimizu-Sasamata M,et al.Attenuation of delayed neuronal death after mild focal ischemia in mice by inhibition of the caspase family[J].J Cereb Blood Flow Metab,1998,18:238-247.
[31]Kerr J F,Winterford C M,Harrnon B V.Apoptosis:Its significance in cancer and cancer therapy[J].Caneer,1994,73(8):2013-2020.
[32]Nicholson D W,Thomberry N A.Caspases:killer Proteases[J].Reviews,1997,22:299-306.
[33]Wang J,Zhen L,Klug M G,et al.Involvement of caspase 3- and 8- like Proteases in ceramide-induced apoptosis of cardiomyocytes[J].J Card Fail.2000,6(3):243-249.
[34]田华,孙婷.半胱氨酸天冬氨酸蛋白酶3抑制剂Ac-DEVD-CHO对心力衰竭大鼠心肌细胞凋亡和心室重构的影响.中国临床康复[J],2005,9(31)96-97.
[35]Philipp S,Pagel I,Hohnel K,et al.Regulation of caspase-3 and Fas in pressure overload-induced left ventricular dysfunction[J].Eur J Heart Fail 2004,287(1):845-851.
[36]孙阳,赵轩.细胞凋亡和PCNA在早孕绒毛滋养细胞及蜕膜中的表达与自 然流产的关系[J].现代妇产科进展,2002,11(2):116-118.
[37]Hammes HP,Wellensiek B,Kloting I,et al.The relationship of glycaemic level to advanced glycation end-product(AGE) accumulaition and retinal pathology in the spontaneous diabetic hamster[J].Diabetologia,1998,41:165-170.
[38]陈百华,姜德咏,唐罗生.半胱氨酸天冬氨酸蛋白酶在视网膜毛细血管周细胞凋亡中的活性及意义[J].中华眼科杂质,2007,43(5),393-396.
[39]Segura I,Serrano A,Buitrago GG,et al.Inhibition of programmed cell death impairs in vitro vascular-like structure formation and reduces in vivo angiogenesis[J].FASEB Journal,2002,16(8):833-841.
[40]萧东 丁健.肿瘤新生血管生成抑制剂的研究进展[J].中国新药杂志,2001,10(4):248-250.
[41]孙惠川 汤钊猷 王鲁等.肝癌诱导新生血管形成及其干预治疗[J].中华普通外科杂志,2001,16(4):216-218.
[42]Carsten S,Sieghart S,Peifeng C,et al.Caspase inhibition activates HIV in latently infected cells.ROLE OF TUMOR NECROSIS FACTOR RECEPTOR 1AND CD95[J].J.Biol.Chem.,2002,277(18):15459-15464.
[43]Deveraux QL,Stennicke HR,Salvesen GS,et al.Endogenous inhibitors of caspases[J].J.Clin.Immunol.,1999,19(6):388-398.
[44]Seve M,Chimienti F,Favier A.Role of intracellular zinc in programmed cell death[J].Pathol Biol(Paris),2002,50(3):212-221.
[45]Lambert JC,Zhou Z,Kang YJ.Supp ression of Fas-mediated signaling pathway is involved in zinc inhibition of ethanol-induced liver apoptosis[J].Exp.Biol.Med.(Maywood),2003,228(4):406-412.
[46]Sliskovic I,Mutus B.Reversible inhibition of Caspase-3 activity by iron(Ⅲ).Potential role in physiological control of apoptosis[J].FEBS Letters,2006,580(9):2233-2237.
[47]任晓,李韶菁,董悦生,等.微生物来源的卡斯帕酶-3抑制剂F03ZA-673A 的研究[J].中国药学杂志,2007,42(12):907-909.
[48]Zech B,Kohl R,von Knethen A,et al.Nitric oxide do-nors inhibit formation of the Apaf-1 /Caspase-9 apoptosome andactivation of Caspase [J]. Biochem. J., 2003, 371 (Pt3): 1055-1064.
[49] Guohua Zhong, Meiying Hu, XiaoyiWei, et al. Grayanane Diterpenoids from the Flowers of Rhododendron molle with Cytotoxic Activity against a Spodoptera frugiperda Cell Line [J]. Journal of Natural Products, 2005, 68 (6): 924-926.
[50] Gunasekera S P, McCarthy P J, Longley R E, et al. A new enzyme inhibitor from a deep-water Caribbean sponge of the genus Batzella [J]. J. Nat. Prod., 1999, 62(1):173-175.
[51] Gunasekera S P, McCarthy P J, Longley R E, et al. Secobatzellines A and B, two new enzyme inhibitors from a deep-water Caribbean sponge of the genus Batzella [J]. J. N at. Prod., 1999, 62 (8): 1208-1211.
[52] Han Y, Giroux A, Grimm E. L, et al. Discovery of novel aspartyl ketone dipeptides as potent and selective caspase-3 inhibitors [J]. Bioorg. Med. Chem. Lett., 2004,14, 805.
[53] Choong I. C, Lew W., Lee D., et al. Identification of potent and selective small-molecule inhibitors of caspase-3 through the use of extended tethering and structure-based drug design [J]. J. Med. Chem., 2002,45, 5005.
[54] Linton S. D., Karanewsky D. S., Ternansky R. J., et al., Acyl peptides as reversible caspase inhibitors. Part 1: Initial lead optimization [J]. Bioorg. Med. Chem. Lett., 2002,12,2969.
[55] Han B. H., Xu D., Choi J., et al., Selective, reversible caspase-3 inhibitor is neuroprotective and reveals distinct pathways of cell death after neonatal hypoxia-ischemia brain injury [J]. J. Biol. Chem., 2002,277, 30128.
[56] Nuttall ME, Nadeau DP, Fisher PW, et al. J Orthop Res , 2000 ,18(3): 56-63.
[57] PG Ekert, J Silke, D L Vaux, et al. Caspase inhibitors [J]. Cell Death Differ, 1999, 6(11): 1081-1086.
[58] Swe M, Sit KH. Z-VAD-FMK and DEVD-CHO induced late mitosis arrest and apoptotic expressions [J]. Apoptosis, 2000, 5 (1): 29 - 36.
[59] Yoshimori A, Sakai J, Sunaga S, Kobayashi T, Takahashi S, Okita N, Takasawa R., Tanuma S. BMC Pharmacol. 2007, 7, 8.
[60] David R. Goode, Anil K. Sharma, and Paul J. Hergenrother. Using Peptidic Inhibitors to Systematically Probe the S1' Site of Caspase-3 and Caspase-7[J]. Org. Lett. 2005, 7, 3529-3532.
[61] Nicola Micale, Rajendran Vairagoundar, et al. Design and synthesis of a potent and selective peptidomimetic inhibitor of caspase-3 [J]. J Med Chem, 2004,47, 6455-6458。
[62] Joseph W. Becker, Jennifer Rotonda, et al. Reducing the Peptidyl Features of Caspase-3 Inhibitors: A Structural Analysis [J]. J Med Chem, 2004,47, 2466-2474.
[63] Dennis Lee, Scott A. Long, et al. Potent and Selective Nonpeptide Inhibitors of Caspases 3 and 7 Inhibit Apoptosis and Maintain Cell Functionality [J]. J Biol Chem, 2000,275(21), 16007-16014.
[64] Kim E S , Yoo S E , Yi K Y, et al. Design, Syntheses and biological evaluations of nonpeptidic caspase 3 Inhibitors [J]. Bull Korean Chem Soc, 2002,23 (7): 1003.
[65] Chen Y, Zhang Y. Design, synthesis, and biological evaluation of isoquinoline-1,3,4-trione derivatives as potent caspase-3 inhibitors [J]. J Med Chem, 2006,49,1613-1623.
[66] Jacobs R T, Folmer J, Simpson T R, et al. Preparation of 2-arylamino-4-quinazolinols as inhibitors of cleavage of protein substrates by caspase-3 [P]. WO: 0121598, 2001-03-29.
[67] Isabel E, Black W C, Bayly C I, et al. Nicotinyl aspartyl ketones as inhibitors of caspase-3 [J]. Bioorg Med Chem Lett, 2003,13(13): 2137.
[68] Choong IC , Lew W, Lee D , et al. Identification of potent and selective small-molecule inhibitors of caspase-3 through the use of extended tethering and structure-based drug design [J]. J Med Chem, 2002 ,45 (23): 5005.
[69] Allen D A, Pham P, Choong I C, et al. Identification of potent and novel Small-molecule inhibitors of caspase-3 [J]. Bioorg Med Chem Lett, 2003,13 (21):3651.
[70]Junichi S,Atsushi Y,Yasuyo N,et al.Structure-based discovery of a novel non-peptidic small molecular inhibitor of caspase-3[J].Bioorg.Med.Chem.,2008,16,4854.
[71]Dennis L,Scott A.L,Jeffrey H.M,et al.Potent and selective nonpeptide inhibitors of caspase-3 and caspase-7[J].J Med Chem,2001,44:2015-2026.
[72]Xue-Qing Yu,Charlie Changli Xue,Zhi-Wei Zhou,et al.Tanshinone ⅡB,A Primary Active Constituent from Salvia miltiorrhiza,exerts Neuroprotective Effect via Inhibition of Neuronal Apoptosis In Vitro[J].Phytother.Res.2008,22,846-850.
[73]Jie Gao,Guoqing Yang,Rongbiao Pi,et al.Tanshinone ⅡA protects neonatal rat cardiomyocytes from adriamycin-induced apoptosis[J].Translational Research.2008,151(2),79-87.
[74]Ji Young Kim,Kyoung Mi Kim,Ji-Xing Nan.Induction of Apoptosis by Tanshinone Ⅰ via Cytochrome c Release in Activated Hepatic Stellate Cells[J].Pharmacology & Toxicology 2003,92,195-200.
[75]Domschke G,J.Prakt.Chem,1966,144.
[76]Mosmann T.Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays[J].J.Immunol.Methods 1983,5,55-63.
[2]Osman A,Ismail B,Alper K,et al.Therapeutic efficacy of Ac-DMQD-CHO,a caspase 3 inhibitor,for rat spinal cord injury[J].J.Clin.Neurosci,2008,15,672.
[3]Junichi S,Atsushi Y,Yasuyo N,et al.Structure-based discovery of a novel non-peptidic small molecular inhibitor of caspase-3[J].Bioorg.Med.Chem.,2008,16,4854.
[4]Zhang Y,Center D M,Wu D M,et al.processing and activation of the pro-interleukin-1β by caspase-3[J].J Biol Chem,1998,273(2):1144-1148.
[5]熊世勤,来锡华.Caspase激活与调控的分子机制[J].生物化学与生物物理进展,2002,27(10):33-36。
[6]王筱冰,张小翠,夏妙红,等.Caspase的活化机制[J].现代生物医学进展,2006,6(3):53-55。
[7]Chang HY et al.Proteases for Cell Suicide:Function and Regulation of Caspase.Microbiol[J].Mol.Biol.Rev,2000,64:821-846.
[8]Thornberry N.A,Rano T.A,Peterson E.P,et al.,A combinatorial approach defines specifieities of members of the caspase family and granzyme B.Functional relationships established for key mediators of apoptosis[J].J.Biol.Chem,1997,272,17907.
[9]Carcia-Calvo M,Peterson E.P,Rasper D.M,et al.Purification and catalytic properties of human caspase family members[J].Cell Death Differ,1999,6,362.
[10]Pistritto G.,Jost M,Srinivasula S.M,et al.Expression and transcriptional regulation of easpase-14 in simple and complex epithelia[J].Cell Death Differ,2002,9,995.
[11]Denault J.B,Salvesen G.S,Caspases:keys in the ignition of cell death[J].Chem.Rev,2002,102,4489
[12]O'Brien T,Lee D.Prospects for caspase inhibitors[J].Mini Rev.Med.Chem,2004,4,153.
[13]Meier P,Finch A,Evan G,Apoptosis in development[J].Nature,2000,407,796.
[14]Armstrong R.C,Aia T.J,Hoang K.D,et al.Activation of the CED3/ICE-related protease CPP32 in cerebellar granule neurons undergoing apoptosis but not necrosis[J].J.Neurosci.,1997,17,553.
[15]Yuan J,Shaham J,Ledoux S,et al.The C.elegans cell death gene ted-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme[J].Cell,1993,75,641.
[16]易成蜡,陈安民.脊柱损伤的细胞凋亡及其调控.中华创伤杂质[J].2003;19(3):190-192.
[17]刘旭,徐平.心肌缺血-再灌注损伤与细胞凋亡[J].中华实用中西医杂志.2005,18(18):981-983.
[18]Saraste A,et al.Apoptosis in human acute myocardial infarction.Circulation[J],1997,95:320-323.
[19]马永刚,刘世清,彭昊,等.硫酸镁对实验性脊髓损伤的保护作用[J].医学研究生学报,2002;15(6):490-492.
[20]Yan Zhang,Cynthia Goodyer,and Andren Le Blanc.Selective andprotracted apoptosis in human primary neurons microinjection withactive caspase-3,-6,-7and -8[J].J Neurosci,2000,20:8384-8389
[21]Cryns V,Yuan J.Protease to die for[J].Genes Dev,1998,12(11):1551-1570.
[22]Communal C,Sumandea M,De Tombe P,et al.Functional consequences of caspase activation in cardiac myocytes[J].Proc Natl Acad Sci USA,2002,99(9):6252-6256.
[23]Enari M,Sakahira H,Yokoyama H,et al.A caspase-activated DNase that degrades DNA during apoptosis,and its inhibitor ICAD[J].Nature,1998,391:43.
[24]Xiao F,Widlak P,Garrard WT.Engineered apoptotic nucleases for chromatin research[J].Nucleic Acids Res,2007,35(13):e93.
[25]Basnakian AG,Ueda N,Hong X,et al.Ceramide synthase is essential for endonuclease-mediated death of renal tubular epithelial cells induced by hypoxia-reoxygenation[J].Am J Physiol Renal Physiol,2005,288(2):F308-314.
[26]Ye H,Cande C,Stephanou NC,et al.DNA binding is required for apoptogenic action of apoptosis inducing factor[J].Nat Struct Biol,2002,9:680-684.
[27]王筱冰,张小翠,夏妙红,等.Caspase的活化机制[J].现代生物医学进展,2006,6(3):53-55.
[28]Kukhta VK,Marozldna NV,Sokolchik IG,et al.Molecular mechanisms of apoptosis[J].Ukr Biokhim Zh,2003,75(6):5-9.
[29]Diep QN,EI Mabrouk M,Yue P,et al.Effect of AT(1) receptor blockade on cardiac apoptosis in angiotensin-induced hypertension[J].Am J Physiol Heart Circ Physiol,2002,282(5):H1635-1641.
[30]Endres M,Namura S,Shimizu-Sasamata M,et al.Attenuation of delayed neuronal death after mild focal ischemia in mice by inhibition of the caspase family[J].J Cereb Blood Flow Metab,1998,18:238-247.
[31]Kerr J F,Winterford C M,Harrnon B V.Apoptosis:Its significance in cancer and cancer therapy[J].Caneer,1994,73(8):2013-2020.
[32]Nicholson D W,Thomberry N A.Caspases:killer Proteases[J].Reviews,1997,22:299-306.
[33]Wang J,Zhen L,Klug M G,et al.Involvement of caspase 3- and 8- like Proteases in ceramide-induced apoptosis of cardiomyocytes[J].J Card Fail.2000,6(3):243-249.
[34]田华,孙婷.半胱氨酸天冬氨酸蛋白酶3抑制剂Ac-DEVD-CHO对心力衰竭大鼠心肌细胞凋亡和心室重构的影响.中国临床康复[J],2005,9(31)96-97.
[35]Philipp S,Pagel I,Hohnel K,et al.Regulation of caspase-3 and Fas in pressure overload-induced left ventricular dysfunction[J].Eur J Heart Fail 2004,287(1):845-851.
[36]孙阳,赵轩.细胞凋亡和PCNA在早孕绒毛滋养细胞及蜕膜中的表达与自 然流产的关系[J].现代妇产科进展,2002,11(2):116-118.
[37]Hammes HP,Wellensiek B,Kloting I,et al.The relationship of glycaemic level to advanced glycation end-product(AGE) accumulaition and retinal pathology in the spontaneous diabetic hamster[J].Diabetologia,1998,41:165-170.
[38]陈百华,姜德咏,唐罗生.半胱氨酸天冬氨酸蛋白酶在视网膜毛细血管周细胞凋亡中的活性及意义[J].中华眼科杂质,2007,43(5),393-396.
[39]Segura I,Serrano A,Buitrago GG,et al.Inhibition of programmed cell death impairs in vitro vascular-like structure formation and reduces in vivo angiogenesis[J].FASEB Journal,2002,16(8):833-841.
[40]萧东 丁健.肿瘤新生血管生成抑制剂的研究进展[J].中国新药杂志,2001,10(4):248-250.
[41]孙惠川 汤钊猷 王鲁等.肝癌诱导新生血管形成及其干预治疗[J].中华普通外科杂志,2001,16(4):216-218.
[42]Carsten S,Sieghart S,Peifeng C,et al.Caspase inhibition activates HIV in latently infected cells.ROLE OF TUMOR NECROSIS FACTOR RECEPTOR 1AND CD95[J].J.Biol.Chem.,2002,277(18):15459-15464.
[43]Deveraux QL,Stennicke HR,Salvesen GS,et al.Endogenous inhibitors of caspases[J].J.Clin.Immunol.,1999,19(6):388-398.
[44]Seve M,Chimienti F,Favier A.Role of intracellular zinc in programmed cell death[J].Pathol Biol(Paris),2002,50(3):212-221.
[45]Lambert JC,Zhou Z,Kang YJ.Supp ression of Fas-mediated signaling pathway is involved in zinc inhibition of ethanol-induced liver apoptosis[J].Exp.Biol.Med.(Maywood),2003,228(4):406-412.
[46]Sliskovic I,Mutus B.Reversible inhibition of Caspase-3 activity by iron(Ⅲ).Potential role in physiological control of apoptosis[J].FEBS Letters,2006,580(9):2233-2237.
[47]任晓,李韶菁,董悦生,等.微生物来源的卡斯帕酶-3抑制剂F03ZA-673A 的研究[J].中国药学杂志,2007,42(12):907-909.
[48]Zech B,Kohl R,von Knethen A,et al.Nitric oxide do-nors inhibit formation of the Apaf-1 /Caspase-9 apoptosome andactivation of Caspase [J]. Biochem. J., 2003, 371 (Pt3): 1055-1064.
[49] Guohua Zhong, Meiying Hu, XiaoyiWei, et al. Grayanane Diterpenoids from the Flowers of Rhododendron molle with Cytotoxic Activity against a Spodoptera frugiperda Cell Line [J]. Journal of Natural Products, 2005, 68 (6): 924-926.
[50] Gunasekera S P, McCarthy P J, Longley R E, et al. A new enzyme inhibitor from a deep-water Caribbean sponge of the genus Batzella [J]. J. Nat. Prod., 1999, 62(1):173-175.
[51] Gunasekera S P, McCarthy P J, Longley R E, et al. Secobatzellines A and B, two new enzyme inhibitors from a deep-water Caribbean sponge of the genus Batzella [J]. J. N at. Prod., 1999, 62 (8): 1208-1211.
[52] Han Y, Giroux A, Grimm E. L, et al. Discovery of novel aspartyl ketone dipeptides as potent and selective caspase-3 inhibitors [J]. Bioorg. Med. Chem. Lett., 2004,14, 805.
[53] Choong I. C, Lew W., Lee D., et al. Identification of potent and selective small-molecule inhibitors of caspase-3 through the use of extended tethering and structure-based drug design [J]. J. Med. Chem., 2002,45, 5005.
[54] Linton S. D., Karanewsky D. S., Ternansky R. J., et al., Acyl peptides as reversible caspase inhibitors. Part 1: Initial lead optimization [J]. Bioorg. Med. Chem. Lett., 2002,12,2969.
[55] Han B. H., Xu D., Choi J., et al., Selective, reversible caspase-3 inhibitor is neuroprotective and reveals distinct pathways of cell death after neonatal hypoxia-ischemia brain injury [J]. J. Biol. Chem., 2002,277, 30128.
[56] Nuttall ME, Nadeau DP, Fisher PW, et al. J Orthop Res , 2000 ,18(3): 56-63.
[57] PG Ekert, J Silke, D L Vaux, et al. Caspase inhibitors [J]. Cell Death Differ, 1999, 6(11): 1081-1086.
[58] Swe M, Sit KH. Z-VAD-FMK and DEVD-CHO induced late mitosis arrest and apoptotic expressions [J]. Apoptosis, 2000, 5 (1): 29 - 36.
[59] Yoshimori A, Sakai J, Sunaga S, Kobayashi T, Takahashi S, Okita N, Takasawa R., Tanuma S. BMC Pharmacol. 2007, 7, 8.
[60] David R. Goode, Anil K. Sharma, and Paul J. Hergenrother. Using Peptidic Inhibitors to Systematically Probe the S1' Site of Caspase-3 and Caspase-7[J]. Org. Lett. 2005, 7, 3529-3532.
[61] Nicola Micale, Rajendran Vairagoundar, et al. Design and synthesis of a potent and selective peptidomimetic inhibitor of caspase-3 [J]. J Med Chem, 2004,47, 6455-6458。
[62] Joseph W. Becker, Jennifer Rotonda, et al. Reducing the Peptidyl Features of Caspase-3 Inhibitors: A Structural Analysis [J]. J Med Chem, 2004,47, 2466-2474.
[63] Dennis Lee, Scott A. Long, et al. Potent and Selective Nonpeptide Inhibitors of Caspases 3 and 7 Inhibit Apoptosis and Maintain Cell Functionality [J]. J Biol Chem, 2000,275(21), 16007-16014.
[64] Kim E S , Yoo S E , Yi K Y, et al. Design, Syntheses and biological evaluations of nonpeptidic caspase 3 Inhibitors [J]. Bull Korean Chem Soc, 2002,23 (7): 1003.
[65] Chen Y, Zhang Y. Design, synthesis, and biological evaluation of isoquinoline-1,3,4-trione derivatives as potent caspase-3 inhibitors [J]. J Med Chem, 2006,49,1613-1623.
[66] Jacobs R T, Folmer J, Simpson T R, et al. Preparation of 2-arylamino-4-quinazolinols as inhibitors of cleavage of protein substrates by caspase-3 [P]. WO: 0121598, 2001-03-29.
[67] Isabel E, Black W C, Bayly C I, et al. Nicotinyl aspartyl ketones as inhibitors of caspase-3 [J]. Bioorg Med Chem Lett, 2003,13(13): 2137.
[68] Choong IC , Lew W, Lee D , et al. Identification of potent and selective small-molecule inhibitors of caspase-3 through the use of extended tethering and structure-based drug design [J]. J Med Chem, 2002 ,45 (23): 5005.
[69] Allen D A, Pham P, Choong I C, et al. Identification of potent and novel Small-molecule inhibitors of caspase-3 [J]. Bioorg Med Chem Lett, 2003,13 (21):3651.
[70]Junichi S,Atsushi Y,Yasuyo N,et al.Structure-based discovery of a novel non-peptidic small molecular inhibitor of caspase-3[J].Bioorg.Med.Chem.,2008,16,4854.
[71]Dennis L,Scott A.L,Jeffrey H.M,et al.Potent and selective nonpeptide inhibitors of caspase-3 and caspase-7[J].J Med Chem,2001,44:2015-2026.
[72]Xue-Qing Yu,Charlie Changli Xue,Zhi-Wei Zhou,et al.Tanshinone ⅡB,A Primary Active Constituent from Salvia miltiorrhiza,exerts Neuroprotective Effect via Inhibition of Neuronal Apoptosis In Vitro[J].Phytother.Res.2008,22,846-850.
[73]Jie Gao,Guoqing Yang,Rongbiao Pi,et al.Tanshinone ⅡA protects neonatal rat cardiomyocytes from adriamycin-induced apoptosis[J].Translational Research.2008,151(2),79-87.
[74]Ji Young Kim,Kyoung Mi Kim,Ji-Xing Nan.Induction of Apoptosis by Tanshinone Ⅰ via Cytochrome c Release in Activated Hepatic Stellate Cells[J].Pharmacology & Toxicology 2003,92,195-200.
[75]Domschke G,J.Prakt.Chem,1966,144.
[76]Mosmann T.Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays[J].J.Immunol.Methods 1983,5,55-63.