Designing a Promotor for a Novel Target Site Identified in Caspases for Initiating Apoptosis in Cancer Cells

Caspases are enzymes that can cleave other proteins and control normal and abnormal cell death. Cancer cells generally lack apoptosis. In this research work, a computational approach has been adopted to design a promotor that targets the inactivated caspases particularly Procaspase3 or caspase7, which are the effector caspases that cleave the downstream substrates like laminA, ICAD and PARP. Out of the 38 anticarcinomic compounds selected for the analysis, some of them are found to have positive charged substituents similar to the known drug; PAC1, which cleaves the safety catch mode that blocks the IETD active site. Site specific interactions of the proteins with these ligands were performed. From the interaction analysis, it was found that 3 compounds; Choline, Glaziovine, Dasatinib can effectively target caspases and activate them. It has been suggested that these compounds favor the activation of the effector caspase proteins, thereby giving a better option in cancer therapy.

Designing a Promotor for a Novel Target Site Identified in Caspases for Initiating Apoptosis in Cancer Cells Mala S Kumar, K.L Lainu, V Aghila, Dhanya Purushothaman, K Varun Gopal, P.K Krishnan Namboori, and Vrinda Harishankar Computational Chemistry Group, Computational Engineering and Networking, Amrita Vishwa Vidyapeetham University, Ettimadai, Coimbatore-641 105, India ammasmaalooty@gmail.com, n_krishnan@cb.amrita.edu, varungopal19@gmail.com Abstract Caspases are enzymes that can cleave other proteins and control normal and abnormal cell death Cancer cells generally lack apoptosis In this research work, a computational approach has been adopted to design a promotor that targets the inactivated caspases particularly Pro-caspase-3 or caspase-7, which are the effector caspases that cleave the downstream substrates like lamin-A, ICAD and PARP Out of the 38 anti-carcinomic compounds selected for the analysis, some of them are found to have positive charged substituents similar to the known drug; PAC1, which cleaves the safety catch mode that blocks the IETD active site Site specific interactions of the proteins with these ligands were performed From the interaction analysis, it was found that compounds; Choline, Glaziovine, Dasatinib can effectively target caspases and activate them It has been suggested that these compounds favor the activation of the effector caspase proteins, thereby giving a better option in cancer therapy Keywords: Apoptosis, caspases, docking, anti-cancer drugs, cancer Introduction The normal cell death process in human body is the deletion of old and damaged cells and replacement of these with healthy new ones When cells are prone to damage, the body responds and inflames the area by rushing blood cells over there to clean up the mess The damaged cells are trapped and are made ready for quick and clean death When given a signal, the cell will rearrange its structure and fragments itself into small tiny pieces that are readily consumed by neighboring cells This process of programmed, controlled death is called apoptosis or programmed cell death [1] Caspases play a major role in the regulation and execution of apoptotic cell death Caspases, a family of cysteinyl aspartate–a specific protease, are synthesized as zymogens with a prodomain of variable length followed by a large subunit (p20) and a small subunit (p10) that are separated by an intersubunit linker and an N-terminal prodomain Depending on the structure of the prodomain and its function, caspases are typically divided into major groups -inflammatory caspases (group I-Caspase-1, V V Das, R Vijaykumar et al (Eds.): ICT 2010, CCIS 101, pp 62–67, 2010 © Springer-Verlag Berlin Heidelberg 2010 Designing a Promotor for a Novel Target Site Identified in Caspases 63 4, 5), initiator caspases (group II-Caspase-2, 8, 9, 10), and effector caspases (group III-Caspase-3, 6, 7) [2] The active site contains a reactive cysteine and three basic amino acids (2 arginines and a glutamate) that recognize the aspartate in the protein that is cleaved These zymogens are floppy and these four aminoacids are not assembled into a tight active site When the caspase is activated by making a few strategic cuts in the protein chain, the active site can form the proper conformation The cysteine is bound to the target protein chain and the aspartate is nestled inside the basic aminoacids Cleavage of intersubunit linker at ASP175 alone is sufficient for autocatalytic activity Also, to ensure that this zymogen is not prematurely activated, pro-caspase has a tri-aspartic acid (DDD) safety catch that blocks access to the IETD (ile-glu-thr-asp) site of proteolysis This results in the generation of mature active caspases that consist of the heterotetramer p202–p102 Subsequently, active caspases specifically process various substrates that are implicated in apoptosis and inflammation [3] Apoptosis is one of the major defenses against cancer and deadly cancer cells often have mutations that disable their own apoptosis machinery To prevent this inactivation, direct initiation of Pro-Caspase proteins are sufficient because apoptosis starts only when these zymogen proteins are cleaved PAC-1 plays a role to activate procaspase-3 indiscriminately When administered to cancer cells, it signals the cells to autodestruct themselves, by initiating the effector markable protein, Procaspase-3 A small molecule containing substituents that are positively charged at physiological pH (such as the piperazine nitrogen in PAC-1) may directly interact with the triaspartic acid safety catch, thereby inducing the auto activation of procaspase-3 [4] Currently, PAC-1 is the only known chemical compound that has been reported to activate procaspase-3 and thereby promotes apoptosis Studies suggest that various phytochemicals and compounds show anti-carcinomic activity This work aims at investigating the possibility of using a suitable phytochemical as promoter for activating the caspase protein This was done by checking the possibility of the phytochemicals to have a behavior similar to that of PAC-1 Curcumin, a pharmacologically safe compound, shows anti-carcinomic properties and current clinical trials have shown that curcumin can also be treated as a chemotherapeutic and chemopreventive agent [5] Here in this work, 38 different compounds have been taken for the interactional studies Materials and Methods Proteins of human caspase-3, Caspase-6 and Caspase-7 were collected from PDB (Protein Data Bank) [6] Then these molecules were subjected to sequence analysis, structural analysis and computational modeling, in order to characterize them Initially, multiple sequence alignment for the proteins caspase-3, Caspase-6 and Caspase-7 was performed using ClustalW tool [7] It calculates the best match for the selected sequences and helps in identifying the similarities and differences between the proteins The physio-chemical properties of the proteins including instability index, aliphatic index, grand average hydropathy (GRAVY), molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, and the half life of these proteins were found out using ProtParam [8] From the half 64 M.S Kumar et al life period of the proteins, the rate of the reaction has been calculated based on first order kinetics and predicted the fastest activation pathway for apoptosis Active sites of these proteins include families, functional sites, amino acid pattern, signatures and motifs and these were determined using PROSITE [9] RADAR tool [10] has been used for finding the repeated regions in the protein sequences The percentage of secondary structures such as alpha chains, beta sheets and random coils, evolutionary relationship and details about the class, folds, domains and super families were found using ‘Self-Optimized Prediction Method’, SOPMA [11] and SCOP [12] database Sub-cellular location of the proteins was identified using Psort tool [13] All the modeling and simulations were performed using Accelrys Discovery studio [14] with ‘smart minimiser’ algorithm and CHARMm force field The input for the modeling was the PDB file collected from the repository Protein Data Bank The collected protein structures were subjected to geometry optimization and the corresponding minimum interactional potential energy, RMS gradient, van der Waals energy; electrostatic energy and kinetic energy values were computed Most of the structures attained convergence at about 2000 steps of iteration To identify a potential promoter, 38 compounds with anti-carcinomic property have been collected The anti-cancer reported drugs used in this analysis include Hesperetin, Paclitaxel, Choline, Dasatinib, etc and the reported phytochemicals used for the analysis include solasodine, Scilliglaucosidin, Cymarin, Quercetin, Homoharringtonine, Hellebrin, Oleandrigenin, Strophanthidin, Scillarenin, Gitoxigenin, Scilliroside, Betulin, Lapachol, GeranylGeraniol, Bullatacin, Curcumin, Daphnorectin, Glaziovine, Taxol, Rhein, Harringtonine, Eupatorin, Tretitonin, Genestin To identify the interactions between the ligands and the proteins, docking was carried out Initially, docking analysis of the protein procaspase-3 with the known ligand PAC-1 was performed using CDOCKER tool [15] of Discovery Studio CDOCKER is a grid-based molecular docking method that employs CHARMm forcefield The receptor is held rigid while the ligands are allowed to flex during the refinement Each protein has numerous active sites and these are docked with the ligand For each final pose, the CDOCKER energy (interaction energy plus ligand strain) and the interaction energy are noted The poses are sorted by CHARMm energy and the top scoring one which is found to be exothermic (thus favorable for binding) is retained The procedure was repeated with all the 38 compounds which were taken for the analysis ADMET [16] tool in Discovery Studio was used for the prediction of pharmacokinetic properties of the given compounds The results for solubility, absorption, blood brain barrier penetration, and hepatoxicity have been analyzed Results and Discussions The instability index values of Caspase-3, 6, have been calculated and it has been found that they are stable in nature Hydropathicity of these proteins were also calculated From the results, it has been observed that all the proteins are hydrophilic in nature The estimated half life of caspase-3 protein was found to be 2.24 hours and for caspase-7 it was found to be 3.00 hours The rate of reaction for the proteins was Designing a Promotor for a Novel Target Site Identified in Caspases 65 also obtained It has been concluded that the activation of caspase-3 (value of K=0.3) is better than the other two proteins caspase-6 and caspase-7 (value of K=0.2287) The sub-cellular location of all proteins has been identified as cytoplasm Generally, Caspase-3 proteins are highly expressed in the lung, spleen, heart, liver and kidney, moderate levels in the brain and skeletal muscles and low levels in the testis Expression of Caspase-7 proteins is absent in the brain From the SCOP database, it was found that caspase-3, and belong to alpha and beta proteins and are in the ‘Caspase catalytic domain family’ Results from ClustalW tool showed the presence of conserved regions in these proteins The active sites of these proteins have been identified using Prosite tool and can be used as the targets for ligand binding The interactional potential energy of these proteins has been found to be negative The van der Waals energy was found to be negative The RMS gradient value was very close to zero indicating them to be in a stable state Table Interaction Energy of Pro-Caspase with PAC-1 Protein ID 2C1E 2C2O 2C2K 2C2M 2J30 2J33 Site Site -42.82 -47.35 25.68 -36.95 -37.74 -40.55 -40.55 -44.16 0 Site Site Site Site Site 0 33.78 0 0 0 0 0 0 0 0 0 0 0 0 0 The site specific interaction energies of Procaspase-3 with the known ligand PAC1 has been calculated and is given in Table Sites and were found to be the best functional sites for the known drug The same procedure was carried out for the 38 compounds also While analyzing the results, interaction energy of ligands were in the same range as that of the known ligand These ligands were characterized using Pre-ADMET The known drug possesses positive charged groups which are involved in the cleavage of procaspase protein Table Result for Ligand Characterization Ligand Pac-1 Choline Glaziovine Dasatinib No of Positive Charged Groups 1 1 No of Amine Groups 1 Polarizability 45.02 42.18 42.32 46.66 Charge on acceptor Atoms -0.003 -0.003 -0.003 -0.005 Ligand Pac-1 Choline Glaziovine Dasatinib Based on these results, compounds which show positive charged groups were screened and listed in Table and from these, the Dasitinib compound contains the same positive charged substituents as that of PAC-1, called piperazine nitrogen that 66 M.S Kumar et al cleaves the specific site ‘DDD’ which blocks the IETD active site The screened ligands were then subjected to pharmacokinetic analysis (ADMET) and the results have been tabulated in Table Table Results of ADMET Analysis on Screened Compounds Ligands Choline Dasatinib Glaziovine ADMET BBB Level 2 ADMET Absorption Level ADMET Solubility Level 3 An ADMET absorption level zero indicates good absorption, indicates medium absorption and shows very poor absorption level Solubility values 2, 3, indicate low, good and optimal solubility levels respectively and solubility level indicates insolubility BBB levels of 0, 1, and indicate very high, high, medium and low BBB penetration respectively Of the three compounds, Choline and Glaziovine were found to have good absorption properties, while Dasatinib showed optimal absorption Considering solubility, all three compounds were found to exhibit good drug action Choline and Glaziovine were found to exhibit medium BBB penetration while Dasatinib showed a relatively high BBB penetration Conclusion Death receptors activate initiator procaspases-8 when the ligand is bound to them Auto-catalytically cleaved procaspase-8 then initiates executioner caspses-3 or or and leads to apoptosis Inactivation of upstream pathway disrupts the activation of these downstream caspases and leads to uncontrolled growth of cells By calculating the estimated half life period and using law of rate of reaction, activation of caspase-3 is found to be good Current studies state that these have a safety catch mode with aspartate aminoacids (DDD) that blocks the active site Isoleucine-GlutamineThreonine-Aspartate (IETD) These proteins are seen as zymogens, and this safety catch enables procaspase-3 to resist autocatalytic activation Known drug PAC-1 cleaves this DDD aminoacids using a small molecule containing substituents that are positively charged (Piperazine Nitrogen) and activates mature caspase-3, eventually leading to apoptotic cell death 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