This is to prove that the thesis work entitled “Anti-malignant property of Curcin through tyrosine kinase inhibition as evidenced by in-silico study” by K. Cartoon image showing the co-localization of Curcin [RED] and PCI [PINK] in the same domain of EGFR (left).
Introduction
The latex is used for the treatment of tumors; stomach ulcers and wound healing in traditional medicine, the leaves of the plant are used as a hemostatic agent and are also drastic purgative. These ingredients indicate pharmacological properties, such as clotting activity of the latex of Jatropha curcas, showed that the whole latex significantly reduced the clotting time of human blood.
Plants of pharmacological importance
Possible uses of Jatropha curcas plant
Most parts of this plant are used for the treatment of various human and veterinary conditions (Shukla et al., 1982). The latex of Jatrofa contains alkaloids, including jatrophin, jatrofam and curcaine, with anticancer properties (Montanaro et al., 1973). Some compounds (Curcin, Curcacyclin A) with antitumor activities were reported to be found in this plant (Berg et al., 1995).
Antibiotic activity of Jatropha has been observed against organisms including Staphylococcus aureus and Escherichia coli (Thomas et al., 2003). Wound healing from curcin ointment was found to be better than nitrofurazone ointment and propamidine isethionate cream in rats (Nath et al., 1992). The dried latex and chloroform extract of the roots have been reported to possess anti-inflammatory activity (Basu et al., 1994).
A single dose of aqueous suspension of dried latex was effective at a significant level against the acute inflammatory response (Kumar et al., 1994). It was also found that larger particles with uneven shapes were stabilized by the curcain enzyme (Bar et al., 2009).
Pharmacological properties of latex
Molecular modeling studies of the peptides in the latex revealed that the silver ions were first trapped in the core structure of the cyclic structure of the protein and then reduced and stabilized in situ by the amide group of the peptide. Many of these traditional medicinal properties of Jatropha curcas need to be investigated in depth for the marketable therapeutic products along with their toxicological effects. Investigation of the coagulation activity of the latex of Jatropha showed that whole latex significantly reduced the clotting time of human blood.
Curcin, a protein extracted from the seeds and latex of Jatropha curcas, is reported to have antitumor properties (Lin Juan et al., 2003). It is one of the main proteins in the latex of Jatropha curcas, it is similar to ricin &. The anticancer property of curcin has been demonstrated on a gastric cancer cell line and human hepatoma (Juan et al., 2003).
Methanol extracts of Hymenocardia acida root bark, Mangifera indica stem bark and Sida rhombifolia leaves showed cytotoxic activity against 60 human cell lines. In an antiviral screen against AIDS, a methanolic extract of Jatropha curcas was found to induce a moderate cytoprotective effect against HIV in cultured human lymphoblastoid CEM-SS cells (Muanza et al., 1995).
Antitumor action mechanism of latex
The RIPs are RNA N-glycosidases (Barbieri et al., 1993) that inactivate ribosomes by site-specifically cleaving the single N-C glycosidic bond between adenine and ribose at A4324 in the 28s rRNA. The depurination of the specific adenine prevents the elongation factor (EF-2) from binding to the 60S subunit, so RIPs can inhibit protein synthesis. Interest in RIPs arose from their potential medical and therapeutic applications because many of these proteins were found to be more toxic to tumor cells than normal cells (Lin et al., 1970).
Studies have shown that they can be used as toxic components of immunotoxins targeting tumor cells (Bolognesi et. al., 2004), abortifacients (Jin et. al., 1985) and antiviral agents (Au et al., 2000; On the basis of structure of the genes and mature proteins (Van Damme et al., 2001), RIPs are classified into three types. Curcin, a protein isolated from latex of J.curcas, shows sequence similarity in amino acid with RIPs (Chow et al. ., 1990).
It is significant that it can inhibit the proliferation of tumor cells, through the induction of apoptosis and differentiation (Xie et al., 2006) of cancer cells. The aim would be to find a proof of anti-malignancy of the latex protein by in-silico approach.
Targeted Pathways of Cancer
Tools used to perform in-silico study .1 Sequence Alignment Tools
- Structure Validation tools
The main objective of structural bioinformatics is to predict the 3D structure of a protein from its 1D protein sequence. The essence of the procedure is the selection of an appropriate structural model based on the similarity of pairs of sequences. Any errors in the sequence alignment format prevent MODELLER from performing homology modeling.
The program is very specific about the extension names of the file formats used for homology modeling. It was developed by the Computational Structural Biology Group at the Swiss Institute of Bioinformatics (SIB) and the Biozentrum of the University of Basel. Each of the tasks is a checkbox option that can be disabled independently if it has already been done or is considered unnecessary.
The Py part of the software's name refers to its extensibility with the Python programming language. To perform a docking screen, the first prerequisite is a structure of the protein of interest.
Experimental plan for in-silico studies of Curcin
- Protein- Protein Docking
Curcin amino acid sequence was collected from UniProt knowledge base and saved in FASTA format. Multiple sequence alignment was performed in CLUSTAL X to obtain the conserved sequence of Curcin from the pool of related protein sequences collected above. The structures of EGFR, mTOR target proteins were collected from the PDB database and saved in .pdb format.
Protien-Protien binding of the above target proteins to Curcin was performed in HEX and interpretation was made based on the binding energies of all. First of all, the amino acid sequence was searched in NCBI for Curcin and saved in FASTA format. The best model based on QMEAN4 score and Z score was then saved in PDB format.
Further validation of the model was done by the RAMPAGE server using the PDB format as input. Once both receptor and ligand proteins are specified as input, their structures will appear in the window.
Results and Discussion
Sequence Alignment
Homology Modeling
Model no. 1 was selected as the best model by Ramachandran Plot analysis as it has the least number of residues in the marginal region.
Structure minimization and visualization using Chimera and PyMol of proteins under study
After the function of binding of the ligand "Curcin" to the "mTOR" receptor, it is observed that curcin preferentially binds to the opposite side of the binding region of Rapamycin, which is responsible for the inhibition of mTOR in the signal transduction pathway. Discussion: The above observation indicates that Curcin does not have a direct role in the signaling pathway via mTOR. Since the formation of the mTOR - Rapamycin complex is necessary for the inhibition of cell proliferation by arresting the cell in the G1 phase; Curcin's role is not important in this respect.
However, the limitations of the in-silico study very well apply here as the above interaction may not be practically feasible in-vivo. Curcin has also been observed to show a similarity in binding properties to EGFR targeting the same domain of EGFR as does PCI. Discussion: Comparable E-total values suggest that successful binding of Curcin to the EGFR tyrosine kinase domain is energetically possible as in the case of the natural inhibitory protein PCI.
Since the above work is an in vitro study, the predicted protein model of Curcin (proving its efficacy by docking studies) needs to undergo clinical trials to determine its efficacy in the development of a new anti-tumor inhibitor. The purpose of the above work is to serve all these researchers for further studies to help the patients who are currently facing this disease.
MULTIPLE SEQUENCE ALIGNMENT
1ONK_A|PDBID|VERIGA|ZAPODJE ---YERLRLRVTH---QTTGEEYFRFITLLRDYVSSGSFS-NEIPLLRQSTIPVSDAQRFVLVELTNQGQDSVTAAIDVTN-AYVVAYQAGDQSYFLR----DAPRGAETH 99 2RG9_A|PDBID|VERIGA|ZAPODJE ---YERLSLR TVQ---QTTGEEYFSFITLLRDFVSSGSFS-NNIPLLRQSTIPVSEASRFVLVELTNEGGDSITAAIDVTN-LYVVAYQAGQQSYFLK----DAPRGAETQ 99 2VC3_A|PDBID|VERIGA|ZAPODJE ---IFPKQYPIINFTTAG---ATVQSYTNFIRAVRGRLTTGADVRHEIPVLPNRVGLPINQR-FILVELSNHAELSVTLALDVTN-AYVVGYRAGNSAYFFHPDNQEDAEAITHL 107 3BJG_A|PDBID|VERIGA|ZAPODJE ---AIFPKQ YPIINFTTAG---ATVQSYTNFIRAVRGRLTTGADVRHEIPVLPNRVGLPINQR-FILVELSNHAELSVTLALDMTN-AAVVGYRAGNSAYFFHPDNQEDAEAITHL 108 gi|42542983|pdb|1J1M|A - --MIFPKQYPIINFTTAG---ATVQSYTNFIRAVRGRLTTGADVRHEIPVLPNRVGLPINQR-FILVELSNHAELSVTLALDVTN-AYVVGYRAGNSAYFFHPDNQEDAEAITHL 108 3EJ5_X|PDBID|CHAIN|SEQUENCE ---QYPIINFTTAG---ATVQSYTNFIRAVRGRLTTGADVRHE IPVLPNRVGLPINQR-FILVELSNHAELSVTLALDVTN-AYVVGYRAGNSAYFFHPDNQEDAEAITHL 103 gi|40889788|pdb|1UQ4|A ---QYPIINFTTAG---ATVQSYTNFIRAVRGRLTTGADVRHEIPVLPNRVGLPINQR- FILVELSNHAELSVTLALDVTN-AYVVGYRAGNSAYFFHPDNQEDAEAITHL 103 1BRY_Y|PDBID|CHAIN|SEQUENCE ---MDVSFRLSG---ATTTSYGVFIKNLREALPYERKV-YNIPLLRSSISGSGR---YTLLHLTNYADETISVAVDVTN-VYIMGYLAGDVSYFFN--E ASATEAAKFV 96 1BRY_Z|PDBID|CHAIN|SEQUENCE ---MDVSFRLSG---ATTTSYGVFIKNLREALPYERKV-YNIPLLRSSISGSGR-- -YTLLHLTNYADETISVAVDVTN-VYIMGYLAGDVSYFFN--EASATEAAKFV 96 2VS6_A|PDBID|CHAIN|SEQUENCE ---MDVSFRLSG---ATSSSYGVFISNLRKALPNERKL-YDIPLLRSSLPGSQR---YALIHLTNYADETISVAIDVTN-VYIMGYRAGDTSYFFN--E ASATEAAKYV 96 2VS6_B|PDBID|VERIGA|ZAPODJE ---MDVSFRLSG---ATSSSYGVFISNLRKALPNERKL -YDIPLLRSSLPGSQR---YALIHLTNYADETISVAIDVTN-VYIMGYRAGDTSYFFN--EASATEAAKYV 96 2JJR_A|PDBID|CHAIN|SEQUENCE ---DVSFRLSG---ATSSSYGVFISNLRKALPNERKL-YDIPLLRSSLPGSQR---YALIHLTNYADETISVAIDVTN-VYIMG YRAGDTSYFFN--EASATEAAKYV 95 3MRW_A|PDBID|VERIGA|ZAPODJE ---DVSFRLSG ---ADPSSYGMFIKDLRNALPHTEKV-YNIPLLLPSVSGAGR---YLLMHLFNYDGNTITVAVDVTN-VYIMGYLALTTSYFFN--EPAADLASQYV 95 3MRY_A|PDBID|VERIGA|ZAPODJE ---DVSFRLSG---ADPSSYGMFIKDLRNALPHTEKV-YNIPLLLPSVSGAGR---YLLMHLF NYDGNTITVAVDVTN-VYIMGYLALTTSYFHN--EPAADLASQYV 95 3N1D_A|PDBID|VERIGA|ZAPODJE ---DVSFRLSG---ADPSSYGMFIKDLRNALPHTEKV-YNIPLLLPSVSGAGR---YLLMHLFNYDGNTITVAVDVTN-VYIMGYLALTTSYFFN--EPAADLASQYV 95 1MOM_A|PDBID|CHAIN|SEQUENCE ---DVSFRLSG---ADPRSYGMFIKDLRNALPFREKV-YNIPLLLPSVSGAGR- --YLLMHLFNYDGKTITVALDVTN-VYIMGYLADTTSYFFN--EPAAELASQYV 95 2OQA_A|PDBID |VERIGA|ZAPODJE ---DVSFSLSG---SSSTSYSKFIGALRKALPSNGTV-YNITLLLSSASGASR---YTLMKLSNYDGKAITVAIDVTN-VYIMGYLVNSTSYFFN--ESDAKLASQYV 95 2OQA_B|PDBID|VERIGA|ZAPODJE ---DVSFSLSG---SSSTSYSKFIGALRKALRKALPSNGTV -YNITLLLSSASGASR---YTLMKLSNYDGKAITVAIDVTN-VYIMGYLVNSTSYFFN--ESDAKLASQYV 95 gi|209156420|pdb|3BWH|A ---NVRFDLSS---ATSSSYKTFIKNLREALPKDGKV-YDIPVLLSTVMDSRR---FILIDLVNYDGQSITAAIDVLN-VYIVAYSTGTVSYFFQ--QVPA-QAPKLL 94 tr|Q8VYU0|Q8VYU0_9ROSI MKG GKMNLSIMVAAWFCWSCIIFGWASAREIVCPFSSNQNYKAGSTPTLTITYDAAADKKNYAQFIKDLREAFGFSYSS-HEIPVLRATVAPNQK---FIVAKVINVANLEVSLGLNVVN-AYLVGYKVGGTSYFFN--DPESLADAKTY 143 3KTZ_A|PDBID|VERIGA|ZAPODJE ---GLDTVSFSTKG ---ATYITYVNFLNELRVKLKPEGNS-HGIPLLRKKCDDPGKC--FVLVALSNDNGQLAEIAIDVTS-VYVVGYQVRNRSYFFK--DAPDAAYEG-- 97 3KTZ_B|PDBID|VERIGA|ZAPODJE ---GLDTVSFST KG---ATYITYVNFLNELRVKLKPEGNS-HGIPLLRKKCDDPGKC--FVLVALSNDNGQLAEIAIDVTS-VYVVGYQVRNRSYFFK --DAPDAAYEG-- 97 1ONK_B|PDBID|CHAIN|SEQUENCE ---DDVTCSASEP---TVRIVGRNGMRVDVRDDDDFHDGNQ---IQLWPSKSNNDPNQLWTIKRDGTIRSNGSCLTTYGYTAGVYVMIFDCNTAVREATIWQIWDNGTIINP 102 2RG9_B|PDBID|CHAIN|SEQU ENCE ---DDVTCSASEP---IVRIVGRNGMTVDVRDDDDFQDGNQ---IQLWPSKSNNDPNQLWTIKKDGTIRSNGSCLTTYGYTAGVYVMIFDCNTAVREATIWQIWGNGTIINP. 1ONK_A|PDBID|VERIGA|ZAPODJE LFTGTTRSSLPFNGSYPDLERYA-GHRDQIPLGIDQLIQSVTALRFPGGS---TRTQARSILILIQMISEAARFNPILWRYRQYINSGASFLPDVYMLELETSWGQQSTQVQ--HSTDGVFNNPIRLAIPPGNFVTLTNVRDVIASLAIM 243 2RG 9_A|PDBID|VERIGA|ZAPODJE DFTGTTRSSLPFNGSYPDLERYA-GHRDQIPLGIDQLIQSVTALRFPGGS---TRTQARSILILIQMISEAARFNPILWRARQYINSGASFLPDVYMLELETSWGQQSTQVQ--HSTDGVFNNPIRLALPPGNVVTLTNIRDVIASLAIM 243 2VC3_A| PDBID|VERIGA|ZAPODJE FTDVQNRYTFAFGGNYDRLEQLAGNLRENIELGNGPLEEAISALYYYSTGGTQLPTLARSFIICIQMISDAARFQYIEGEMRTRIRYNRRSAPDPSVITLENSWGRLSTAIQ--ESNQGAFASPIQLQRRNGSKFSVYDVSILIPIIALM 255 3BJG_A| PDBID|VERIGA|ZAPODJE FTDVQNRYTFAFGGNYDRLEQLAGNLRENIELGNGPLEEAISALYYYSTGGTQLPTLARSFIICIQMISEAARFQYIEGEMRTRIRYNRRSAPDPSVITLENSWGRLSTAIQ--ESNQGAFASPIQLQRRNGSKFSVYDVSILIPIIALM 256 gi|42542983|pdb|1J 1M|A FTDVQNRYTFAFGGNYDRLEQLAGNLREDIELGNGPLEEAISALYYYSTGGTQLPTLARSFIICIQMISEAARFQYIEGEMRTRIRYNRRSAPDPSVITLENSWGRLSTAIQ--ESNQGAFASPIQLQRRNGSKFSVYDVSILIPIIALM 256 3EJ5_X|PDBID|VERIGA|ZAPODJE FTDVQNRY TFAFGGNYDRLEQLAGNLRENIELGNGPLEEAISALYYYSTGGTQLPTLAISFIICIQMISEAARFQYIEGEMRTRIRYNRRSAPDPSVITLENSWGRLSTAIQ--ESNQGAFASPIQLQRRNGSKFSVYDVSILIPIIALM 251 gi|40889788|pdb|1UQ4|A FTDVQNRYTFAFGGNYDRLEQLAGN LRENIELGNGPLEEAISALYYYSTGGTQLPTLARSFIICIQMISEAARFQYIEGEMRTRIRYNRRSAPDPSVITLENSWGDLSTAIQ--ESNQGAFASPIQLQRRNGSKFSVYDVSILIPIIALM 251 1BRY_Y|PDBID|VERIGA|ZAPODJE FKDAKKKVTLPYSGNYERLQTAAGKIRENIPLGLPALDSAITTLYYYT---ASSAASALLVLIQSTAESARYKFIEQQIGKRVDKTFL--PSLATISLENNWSALSKQIQIASTNNGQFESPVVLIDGNNQRVSITNASARVVTSNIA 239 1BRY_ Z|PDBID|VERIGA|ZAPODJE FKDAKKKVTLPYSGNYERLQTAAGKIRENIPLGLPALDSAITTLYYYT---ASSAASALLVLIQSTAESARYKFIEQQIGKRVDKTFL--PSLATISLENNWSALSKQIQIASTNNGQFESPVVLIDGNNQRVSITNASARVVTSNIA 239 2VS6_A|PDBID |VERIGA|ZAPODJE FKDAMRKVTLPYSGNYERLQTAAGKIRENIPLGLPALDSAITTLFYYN---ANSAASALMVLIQSTSEAARYKFIEQQIGAAVDATFL--PSLAIISLENSWSALSKQIQIASTNNGQFESPVVLINAQNQRVTITNVDAGVVTSNIA 239 2VS6_B| PDBID|VERIGA|ZAPODJE FKDAMRKVTLPYSGNYERLQTAAGKIRENIPLGLPALDSAITTLFYYN---ANSAASALMVLIQSTSEAARYKFIEQQIGAAVDATFL--PSLAIISLENSWSALSKQIQIASTNNGQFESPVVLINAQNQRVTITNVDAGVVTSNIA 239 2JJR_A|PDB ID|VERIGA|ZAPODJE FKDAMRKVTLPYSGNYERLQTAAGKIRENIPLGLPALDSAITTLFYYN---ANSAASALMVLIQSTSEAARYKFIEQQIGKRVDKTFL--PSLAIISLENSWSALSKQIQIASTNNGQFESPVVLINAQNQRVTITNVDAGKVTSDIA 238 3MRW_A|PDBID|CHA V|SEKVENCI FRSARRKITLPYSGNYERLQIAAGKPREKIPIGLPALDTAISTLLHYD---STAAAGALLVLIQTTAEAARFKYIEQQIQERAYRDEV--PSSATISLENSWSGLSKQIQLAQGNNGVFRTPTVLVDSKGNRVQITNVTSNVVTSNIQ 238 3MRY_A|PDBID| VERIGA|ZAPODJE FRSARRKITLPYSGNYDRLQIAAGKPREKIPIGLPALDTAISTLLHYD--STAAAGALLVLIQTTAEAARFKYIEQQIQERAYRDEV--PSSATISLENSWSGLSKQIQLAQGNNGVFRTPTVLVDSKGNRVQITNVTSNVVTSNIQ 238 3N1D_A|PDBID|VERIGA|SEQU ENCE FRSARRKITLPYSGNYERLQIAAGKPREKIPIGLPALDTAISTLLHYD---STAAAGALLVLIQTTAEAARFKYIEQQIQERAYRDEV--PSSATISLENSWSGLSKQIQLAQGNNGVFRTPTVLVDSGGNRVQITNVTSNVVTSNIQ 238 1MOM_A|PDBID|CHAIN|SEQUENCE FRDARRKITLPY SGNYERLQIAAGKPREKIPIGLPALDSAISTLLHYD---STAAAGALLVLIQTTAEAARFKYIEQQIQERAYRDEV--PSLATISLENSWSGLSKQIQLAQGNNGIFRTPIVLVDNKGNRVQITNVTSKVVTSNIQ 238 2OQA_A|PDBID|CHAIN| ZAPOREDJE FAGS-TIVTLPYSGNYEKLQTAAGKIREKIPLGFPALDSAITTLFHYD---STAAAAAFLVIIQTTAESSRFKYIEGQIIMRISKNGV--PSLATISLENEWSALSKQIQLAQTNNGTFKTPVVIMDAGGQRVEIGNVGSKVVTKNIQ 237 2OQA_B|PDBID|CHAIN|SEQU ENCE FAGS-TIVTLPYSGNYEKLQTAAGKIREKIPLGFPALDSAITTLFHYD---STAAAAAFLVIIQTTAESSRFKYIEGQIIMRISKNGV--PSLATISLENEWSALSKQIQLAQTNNGTFKTPVVIMDAGGQRVEIGNVGSKVVTKNIQ 237 gi|209156420|pdb|3BWH|A FKGT-QQRTLPYTGNYENLQTAAKKLRENIELGLPALDSAITTLFHYN---AEAAASALLVLIQTTSEAARFRYIELQIANNVGTKFK--PSQTIISLENNWSALSKQIQIAKNKNGQFETPVILIDPQGNRVQITNVTSNVVTQNIQ 236 tr|Q8VYU0|Q8VYU0_9ROSI LFTDTKQQTLSFTGSYADFLSRANVHREDVDLGVQALDNYIYTLEKSSK----PADIAKPLVGFIEMVPEAARFKYIEKVLSQISKTLR--PGGDIISLENNWGDLSYQIQKCVN--GVFLKPVQLQRENYTNILVNNVTQVAGVMGVL 2 85 3KTZ_A|PDBID|CHAIN|SEQUENCE LFKNTIKTRLHFGGSYPSLEGEK-AYRETTDLGIEPLRIGIKKLDENAIDNYKPTEIASSLLVVIQMVSEAARFTFIENQIRNNFQQRIR--PANNTISLENKWGKLSFQIRTSGAN-GMFSEAVELERANGKKYYVTAVDQVKPKIALL 243 3KTZ_B |PDBID|CHAIN|SEQUENCE LFKNTIKTRLHFGGSYPSLEGEK-AYRETTDLGIEPLRIGIKKLDENAIDNYKPTEIASSLLVVIQMVSEAARFTFIENQIRNNFQQRIR--PANNTISLENKWGKLSFQIRTSGAN-GMFSEAVELERANGKKYYVTAVDQVKPKIALL 243 1ONK_B|PDBID|VERIGA|ZAPODJE RSNLVLAASSGIKGTTLTVQTLDYTLGQGWLAGNDTAPREVTIYGFRDLC---MESNGGSVWVETCDSSQKNQKWALYGDGSIRPKQNQDQCLTSGRDSVSTVINIVSCSGASGSQRWVFTNEGAILNLKNGLAMDVAQANPKLRRIIIY 249 2RG9 _B|PDBID|VERIGA|ZAPODJE RSNLVLAASSGIKGTTLTVQTLDYTLGQGWLAGNDTAPREVTIYGFRDLC---MESNGGSVWVETCTIGQENQRWALYGDGSIRPKQNQSQCLTNGRDSVSTVINIVSCSAGSSGQRWVFTNEGAILNLKNGLAMDVAQANPKLRRIIIY.
