Design, Synthesis, Activity And Molecular Docking Study Of Novel Nonpeptide Small-Molecule Inhibitors Of Caspase-3

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.Tanshinoneâ…¡A(IC₅₀= 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 tanshinoneâ…¡A,sodium tanshinoneâ…¡A sulfonate (STS) which was a comercially available Chinese medicine,showed much higher inhibitory activity(IC₅₀= 0.36μM) than Tanshinoneâ…¡A.Moreover,4-[bis(4-hydroxyphenyl)methylene]-2,5-cyclohexadien-1-one(rosolic acid) with an IC₅₀ of 0.27μM,was identified as a novel inhibitor of caspase-3.This paper was divided into two parts.Partâ… 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(IC₅₀=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.DesignIn 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.SynthesisThe 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 AssayThe 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(IC₅₀=0.11μM and 0.21μM).Parallel experiments demonstrated that the IC₅₀ 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 StudyMolecular 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.Partâ…¡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(IC₅₀= 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.DesignIn 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.SynthesisPhenolphthalein 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 AssayRosolic 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(IC₅₀= 0.27μM and 0.14μM).Parallel experiments demonstrated that the IC₅₀ 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 StudyMolecular 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 tanshinoneâ…¡A 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.