Synthesis, Docking and Pharmacological Evaluation of Some Tetrapeptides

“SYNTHESIS, DOCKING AND PHARMACOLOGICAL EVALUATION OF SOME TETRAPEPTIDES”

SYNOPSIS FOR

M.PHARM. DISSERTATION

SUBMITTED TO

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES

KARNATAKA

BY

Ms. MAHIMA MALIPEDDI

I M.PHARM

DEPARTMENT OF PHARMACEUTICAL CHEMISTRY

PESCOLLEGE OF PHARMACY

BANGALORE-560 050

(2011-2013)

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1. / Name of the candidate and address / Ms. MAHIMA MALIPEDDI
I. M. PHARM
(PHARMACEUTICAL CHEMISTRY)
PESCOLLEGE OF PHARMACY
HANUMANTHANAGAR
BANGALORE-560 050
PERMANENT ADDRESS:
D/O DR.M.HIMAJA
NO. 706, RAJESWARITOWERS
VIT UNIVERSITY QUARTERS
VELLORE
TAMILNADU-632014
2. / Name of the institution / PESCOLLEGE OF PHARMACY
HANUMANTHANAGAR
B.S.K.1st STAGE
BANGALORE:-560 050
3. / Course of the study / MASTER OF PHARMACY
(PHARMACEUTICAL CHEMISTRY)
4. / Date of Admission / 13th AUGUST 2012
5. / Title of the topic:
“SYNTHESIS, DOCKING AND PHARMACOLOGICAL ACTIVITY OF SOME TETRAPEPTIDES”
Tetrapeptide 1 Tetrapeptide 2
6. / BRIEF RESUME OF THE INTENDED WORK
6.1 Need for the study:
In the past few years, a wide variety of naturally occurring bioactive linear and cyclic peptides have been isolated from animal and plant sources, culture filtrates, marine sponges and tunicates. Many of the peptides are found to exhibit antimicrobial1, antitubercular2, anticancer3, insecticidal4,5, antioxidant6, anthelmintic7, tyrosinase inhibitory activities8.
Most of these peptides exhibit their biological activities through binding to the corresponding acceptor molecules (receptors or enzymes). This characteristic feature can allow bioactive peptides to act as therapeutic agents. Unfortunately the native peptides for clinical applications have been limited because of the intrinsic properties of peptides i.e.,
1. Rapid degradation of peptides by peptidase enzymes leading to metabolic instability which complicates oral delivery of peptides. Passage through the blood brain barrier is an additional problem for peptides which act on the CNS.
2.The inherent flexibility of peptides which leads to different conformational binding to more than one receptor sites may lead to undesirable effects.
Recently small chain linear peptides have exhibited wide varieties of activities like antioxidant, anticancer, antimicrobial etc. These can lead to discovery of peptide-based cost effective drug molecules.
Based on the above facts an attempt will be made to synthesise peptide based cost effective drug molecules.
6.2Review of the literature:
References pertaining to Peptides:
1. Gunnur Dikmen et al9reported that Hypoxia Inducible Factor-1alpha (HIF-1alpha)plays a major role in activating gene transcription and is important for maintaining homeostasis under hypoxic conditions. Since tumors are often in a hypoxic state, HIF-1alpha is a potential target for the development of novel cancer therapeutics. This study was performed to determine the antitumoral efficacy of an antisense HIF-1a inhibitor, RX-0047 on different human cancer cell lines (MDA-MB 231, HME50-T, PC-3, Panc-1 and A549) in vitro.
2. Katsuhiro Kudo et al10 reported three peptides, purified from potato Proteinhydrolysate fractions, possessedantioxidative activities. These three peptides Phe-Gly-Glu-Arg,
Phe-Asp-Arg-ArgandPhe-Gly-Glu-Arg-Arg, exhibit cytotoxic activity.


3. Boger et al11synthesized potent antitumor antibiotics Deoxybouvardin and RA-VII analogs which contain tetrapeptide subunit D-AIa-AIa-NMe-Tyr(OMe)-AIa, substituted by (Gly)4 for Deoxybouvardin and (Gly)3 for RA-VII.


4. Alejandro et al12presented a structured review of the antitumor and cytotoxic properties of marine natural product peptides


5. Norikazu Nishino et al13synthesized cyclic tetrapeptide retrohydroxamicacids and evaluated the products as potential anticancerdrugs.



6. Luke Simmons et al.14isolated Belamide A from the marine cyanobacterium Symploca sp. Belamide A is a highly methylated linear tetrapeptide with structural analogy to the important linear peptides dolastatins and it showed moderate cytotoxicity.

Dolastatin 15 Belamide A
7.Beverly A.Teicher et al15synthesized and found PS-341 to be a potent cytotoxic agent toward MCF-7
human breast carcinoma cells in culture, producing an IC90 of 0.05 mM on 24hr of exposure to the drug.


8.Mona D. Lee et al16found that actinonin antibiotic analogs are the inhibitors of mitochondrial PDF enzyme and act as anticancer agent. Analogs of actinonin were designed and synthesized and they were found to act as tumour suppressive agent.
9.Yuan Yang et al17studied abouthypoxia-inducible factor 1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) which are frequently over expressed in numerous types of cancers and are known to be importantregulators of angiogenesis. The purpose of this study was to evaluate the expression of HIF-1α and VEGF in CRC tissues, and to analyze the association of these two factors with several clinical and pathological characteristics, and patient’s survival.
10.Ryanet al18reported that the transcriptional response to lowered oxygen levels mediated by the hypoxia-inducible transcription factor (HIF-1),a heterodimer consisting of the constitutively expressed aryl hydrocarbon receptor nucleartranslocator (ARNT) and the hypoxic response factor HIF-1α. To study the role of the transcriptional hypoxic responseinvivothey targeted the murine HIF-1α gene. Loss ofHIF-1α in embryonic stem (ES) cells dramatically retards solid tumor growth.
11.Jun Wanet al19reported that HIF-1alpha, which is induced by hypoxia, is the most important regulatory factor of many specific genes that can influence the biological features of tumors. In this study, they tried to elucidate the changes in gene expression profiles of SCLC NCIH446 cells mediated by HIF-1alpha.
12. In-Sung Songet al20 studied about hypoxia-inducible factors (HIFs) which are transcription factors that activate the transcription of target genes involved in crucial aspects of cancer development. Their study investigated the expression of HIFs and their contribution to the regulation of target genes related to angiogenesis and glucose metabolism in gastric cancer.
Wainunuamide21was isolated by Marcel et al in 2005 from the Western Pacific marine sponge Fijian species. Significant anticancer activity was detected in the crude extract of Stylotella aurantium (order Halichondrida; Family Halichondriidae). The sponge was collected in December 1997 at a depth of about 5m from Cakaulevu reef, in the district of Wainunu, in the island of Vanua Levu, FijiIslands. The marine sponge was extracted with a mixture of methanol and dichloromethane (50/50) as eluent. The structural elucidation was done with high resolution ESIMS and 1H, 13C and 2D NMR spectral analysis. The compound was separated and purified by preparative HPLC chromatographic analysis method. All the amino acids in wainunuamide possess L-configuration.
Wainunuamide exhibited strong in vitro cytotoxicity against the A2780 ovarian tumour and K562 leukaemia cancer cell line.

Various tetrapeptides can be derived from the amino acid sequence of Wainunuamide to explore the pharmacophore of the molecule.
6.3 OBJECTIVES OF THE STUDY:

1. To synthesize and characterize some tetrapeptides.

2. The purity and progress of the reactions will be monitored by TLC.
3. The purification of the compounds will be carried out by recrystallization using suitable solvents.

1. To characterize the structures of newly synthesized compounds by Melting Point, IR, 1H-NMR, 13C-NMR and MASS spectra.

1. Pharmacological screening of synthesized tetrapeptides like anticancer, antitubercular, antimicrobial and antioxidant activities.

1. Docking studies of various cancer receptors using Hex and Molegrow softwares.

1. To publish the research work in peer reviewed journals.

Scheme:

7. / MATERIALS AND METHODS:
Chemicals and other reagents will be purchased from standard companies. The progress of the reactions will be monitored by micro thin layer chromatography. The standard protocols will be followed for purification of the products. Structures of the synthesized molecules will be confirmed by the analytical and spectral data. Docking studieswill be carried out using Hex and Molegrow softwares.
7.2 Sources of data
IISC LIBRARY, BANGALORE
PESCP LIBRARY, BANGALORE
RGUHS DIGITAL LIBRARY (Helinet)
CENTRAL COLLEGE LIBRARY, BANGALORE
7.3 Method of collection of data
Chemical Abstracts, Journals like Journal of Cellular Biochemistry, Journal of Functional Foods, Bioorganic and Medicinal Chemistry, European Journal of Cancer, Journal of Clinical Investigation, BMC Cancer, The EMBO Journal, Clinical Cancer Research, Journal of Experimental and Clinical Cancer Research, Experimental and Molecular Research andScience Direct.
7.4 Pharmacological evaluation:

1. Evaluation of anticancer activity will be done using MTT Assay method on HeLa cell lines.
2. Evaluation of antitubercular activity will be done using Alamar Blue method.
3. Evaluation of antimicrobial activity will be done using Cup Plate method or Disc Diffusion method.
4. Evaluation of antioxidant activity will be done using DPPH method.

LIST OF REFERENCES:
1) Alassane Wele, I. Ndoye Y., Zhang J. Paul, Pousset B.L. and Bodo, B (2005). “Glaucacyclopeptide A from the seeds of Annona glauca”, Phytochemistry, Vol. 66, pp. 1154–1157.
2)Yoshioka H., T.Aoki H.,Goko K.,Nakatsu T.,Noda H.,Sakakibara T.,Take, A.,Nagata J.,Abe T.Wakamiya, Shiba T and T.Kaneko(1971). “Chemical Studies on Tuberactinomycin II. The structure of Tuberactinomycin O”, Tetrahedron Lett., Vol. 12, pp. 2043-2044.
3) Himaja, M., Ranjitha J.and Sunil V. Mali (2011).“Synthesis, Docking And Anticancer Activity Studiesof D-Proline-incorporated Wainunuamide”, Journal of chemical Sciences, Vol 124 (5), pp. 1049-1055.
4) Danuta Konopińska, Wieslaw Sobótka, Andrzej Lesicki, Grzegorz Rosiński and Przemyslaw Sujak (1986) “Synthesis of proctolin analogues and their cardioexcitatory effect on cockroach, Periplaneta americana L., and yellow mealworm, Tenebrio molitor L.”, International Journal of Peptide and Protein Research, Vol 27 (6), pp.597-603.
5) M. Himaja, Sanjay Sharma, Poppy Das, Asif Karigar And D. Munirajasekhar, (2013), “Synthesis, Docking And Biological Studies Of The Linear Tetrapeptide Pwpv - A Potent Insecticidal Agent”, International Journal of Pharmacy and Pharmaceutical Sciences, Vol 5, pp 156-159.
6) M. Himaja, Sreekanth K., D. Munirajasekhar, M.V. Ramana and Mukesh Sikarwar (2011). “Computer-aided Design, Synthesis and Antioxidant Activity of Linear Tetrapeptide D-Phe-L-(Ala-Tyr-Val)”,Journal of Pharmacy Research, Vol 4(8), pp. 2581-2583.
7)Rajiv Dahiya, Devender Pathak, Malipeddi Himaja And Sunita Bhatt(2006), “First Total Synthesis And Biological Screeningof Hymenamide E”, Acta Pharmaceutica, Vol56, pp 399-415.
8) Hiroshi Morita, Takashi Kayashita, Hideyuki Kobata, Akira Gonda, Koichi Takeya and Hideji Itokawa (1994), “Pseudostellarins A - C, new tyrosinase inhibitory cyclic peptides from Pseudostellaria heterophylla”, Tetrahedtron, Vol 50 (23), pp 6797–6804.
9) Z. Gunnur Dikmen,Ginelle C. Gellert, Pakize Dogan, Heejeong Yoon, Young Bok Lee,Chang Ho Ahn and Jerry W. Shay(2008) “Invivo and Invitro Effects of a HIF-1α Inhibitor,RX-0047’’, Journal of Cellular Biochemistry, Vol.104,pp.985–994.
10) Katsuhirokudo, Shuichi Onodera,Yasuyuki Takeda, Noureddine Benkeblia, Norio Shiomi(2009)”Antioxidative activities of some peptides isolated from hydrolyzed potato protein extract”, Journal of functional foods, Vol I ,pp.170-176.
11) Dale L. Boger, Jiacheng Zhou, Brian Writer and Paul A. Kitos(1995) “Key analogs of the Tetrapeptide Subunit of RA-VII and Deoxybouvardin”, Bioorganic and Medicinal Chemistry, Vol. 3, No. 12, pp. 1579-1593.
12) Alejandro M.S.Mayera, Kirk R. Gustafson(2008),“Marine pharmacology in 2005–2006: Antitumor and cytotoxic compounds”, European journal of Cancer, Vol. 44, pp. 2357-2387.
13) Norikazu Nishino, Gururaj M. Shivashimpi, Preeti B. Soni, Mohammed P. I. Bhuiyan, Tamaki Kato, Satoko Maeda, Tomonori G. Nishino and Minoru Yoshida(2008),”Interaction of aliphatic cap group in inhibition of histone deacetylases by cyclic tetrapeptides”,Bioorganic and Medicinal Chemistry, Vol. 16, pp. 437-445.
14) T. Luke Simmons Kerry L. McPhail, Eduardo Ortega-Barria, Susan L. Mooberryd and William H. Gerwicka (2006),”Belamide A, a new antimitotic tetrapeptide from a Panamanian marine cyanobacterium”, Tetrahedron Letters, Vol no. 47, pp. 3387–3390.
15) Beverly A.Teicher, Gulshan Ara, Roy Herbst, Vito J. Palombella and Julian Adams(1999) “The ProteasomeInhibitor PS-341 in Cancer Therapy”,Clinical Cancer Research,Vol. 5, pp. 2638-2645.
16) Mona D. Lee,Yuhong She, Michael J. Soskis, Christopher P. Borella, Jeffrey R. Gardner, Paula A. Hayes, Benzon M. Dy, Mark L. Heaney, Mark R. Philips, William G. Bornmann, Francis M. Sirotnak and David A. Scheinberg(2004),“Human mitochondrial peptidedeformylase, a new anticancer target of actinonin-based antibiotics”, The Journal of Clinical Investigation, Vol. 114, No. 8.
17) Yu Yang, Dan Cao, Mei Hou, Yong-song Guan, Ming JiangandHongfeng Gou(2009)”Expression of HIF-1α and VEGF in colorectal cancer:association with clinical outcomes andprognostic implications’’, BMC Cancer, Vol. 9, pp. 432.
18) Heather E. Ryan, Jessica Lo and Randall S. Johnson(1998)”HIF-1a is required for solid tumor formation and embryonic vascularization”, The EMBO Journal,Vol. 17, No.11 pp. 3005–3015.
19) Jun Wan, Jinben Ma, Ju Meiand GenfaShan(2009).’’The effects of HIF-1alpha on gene expression profiles of NCI-H446 human small cell lung cancer cells”, Journal of Experimental and Clinical Cancer Research, Vol. 28, pp. 150.
20) In-Sung Song, Ai-Guo Wang,Sun Young Yoon, Jeong-Min Kim,Joo Heon Kim, Dong-Seok LeeandNam-Soon Kim(2009)’’Regulation of glucose metabolism-related genes and VEGF by HIF-1α and HIF-1β, but not HIF-2α in gastric cancer’’, Experimental and Molecular Medicine, Vol. 41, No. 1, pp. 51-58.

21) Jioji Tabudravu, Linda A. Morris, J.Jantina, Kettenes-van den Bosch, Marcel Jaspars (2001), “Wainunuamide, a histidine-containing proline-rich cyclic heptapeptide isolated from the Fijian marine sponge Stylotella aurantium”, Tetrahedtrn Letters, Vol 42, pp.9273–927

9 / Signature of the candidate : / (MAHIMA MALIPEDDI)
10. / Remarks of the guide: / FORWARDED WITH REQUEST
FOR APPROVAL
11. / Name and designation of:
11.1 GUIDE
11.2 SIGNATURE / Mrs. P.N. KAVITA
Assistant Professor
Department of Pharm Chemistry,
PESCollege of Pharmacy
Bangalore- 560 050
11.3 CO-GUIDE
11.4 SIGNATURE / NOT APPLICABLE
11.5 HEAD OF THE DEPARTMENT
11.6 SIGNATURE / Dr. J. Saravanan
Prof. and HOD
Department of Pharm Chemistry,
PESCollege of Pharmacy
Bangalore- 560 050
12. / 12.1 REMARKS OF THE PRINCIPAL:
12.2 SIGNATURE: Prof. Dr. S. Mohan
Principal & Director,
PESCollege of Pharmacy,
Bangalore-560 050