bioRxiv preprint doi: https://doi.org/10.1101/2020.05.08.083972; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Research Article 2 3 Novel antiproliferative tripeptides block AP-1 transcriptional complex by in silico approach 4 5 6 Ajay Kumar Raj, Jainish Kothari, Sethamma TN Sinchana, Kiran Lokhande, K. V. Swamy, 7 Nilesh Kumar Sharma* 8 9 Cancer and Translational Research Lab, Dr. D.Y. Patil Biotechnology & Bioinformatics 10 Institute, Dr. D.Y. Patil Vidyapeeth, Pune, Maharashtra, India, 411033. 11 12 13 14 15 16 17 *Corresponding author: 18 Dr. Nilesh Kumar Sharma 19 Professor 20 Cancer and Translational Research Lab 21 Department of Biotechnology 22 Dr. D. Y. Patil Biotechnology & Bioinformatics Institute, Pune 23 Dr. D. Y Patil Vidyapeeth Pune, Pune, MH, 411033 24 Email: [email protected] 25 Phone: +91-7219269540 26 27 ORCID ID: 28 Dr. Nilesh Kumar Sharma https://orcid.org/0000-0002-8774-3020 29 30 31 ACKNOWLEDGEMENTS: 32 The authors acknowledge financial support from DST-SERB, Government of India, New Delhi, 33 India (SERB/LS-1028/2013) and Dr. D.Y. Patil Vidyapeeth, Pune, India (DPU/05/01/2016). 34 35 CONFLICT OF INTEREST 36 The authors declare that they have no conflict of interest. 37 38 39 40 41 42 43 44 45 46 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.08.083972; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 ABSTRACT: 2 3 BACKGROUND 4 The complexity and heterogeneity at genetic, epigenetic and microenvironment levels are key 5 attributes of tumors. Genetic heterogeneity encompasses one of key factors at transcriptional 6 gene regulation that promote abnormal proliferation, invasiveness and metastasis. Among 7 various key pro-tumor transcriptional complexes, activating protein-1 (AP-1) transcriptional 8 complex controls the transcriptional expression of key oncogenes in cancer cells. Therefore, an 9 avenue to search for a chemical inhibition approach of the AP-1 transcriptional complex is 10 warranted in cancer therapeutics. 11 12 METHODS 13 To achieve chemical inhibition of AP-1 transcriptional complex, we report novel tripeptides 14 identified from the goat urine DMSO fraction as potential agents that bind to AP-1 responsive 15 TPA element and heterodimer c-Jun:c-Fos. Novel tripeptides enriched GUDF were tested against 16 DNA substrates to assess DNA metabolizing activity. Further, Novel tripeptides enriched GUDF 17 were treated upon HCT-116 cells to estimate the nature of tripeptides entered into the 18 intracellular compartment of HCT-116 cells. Here, we report on a novel methodology that 19 employ VTGE assisted intracellular metabolite purification and is analyzed with the help of LC- 20 HRMS technique. Post purification of intracellular metabolites that included tripeptides of 21 GUDF, these tripeptides from DMSO and GUDF treated HCT-116 cells were subjected to 22 molecular docking and ligand-DNA:AP-1 (PDB ID: 1FOS) interaction study by using 23 bioinformatics tools AutoDock Vina and PyMol. 24 25 RESULTS 26 GUDF enriched with tripeptides and other metabolites show appreciable instability of DNA 27 substrates plasmid and genomic DNA to an extent of 90%. Interestingly, LC-HRMS analysis of 28 intracellular metabolite profiling of GUDF treated HCT-116 cells reveal the appreciable 29 abundance of tripeptides Glu-Glu-Arg, Gly-Arg-Pro, Gln-Lys-Arg, Glu-Glu-Lys, Trp-Trp-Val. 30 On the other hand, DMSO treated HCT-116 cells show the presence of Ser-Trp-Lys, Glu-Glu- 31 Gln, Glu-Glu-Lys, Ser-Leu-Ser. Interestingly, GUDF treated HCT-116 cells show inhibition of 32 proliferation by more than 70%. Among the identified intracellular tripeptides, Glu-Glu-Arg (9.1 33 Kcal/Mol), Gly-Arg-Pro (8.8 Kcal/Mol), and Gln-Lys-Arg (6.8) show a precise and strong 34 binding to heptameric TPA response element 5` TGAGTCA 3` and key amino acid residue 35 within the AP-1 transcriptional complex. 36 37 CONCLUSION 38 In summary, this study suggests the potential of novel tripeptides, those are reported from GUDF 39 intracellularly in HCT-116 cells to destabilize the AP-1 transcriptional complex. Data indicate 40 that cellular arrest in HCT-116 cells treated by GUDF is well supported by the molecular 41 docking observations that destabilization of AP-1 complex is linked to reduced growth and 42 proliferation. 43 44 Keywords: Microbiomes, Metabolites, Tri-peptides Cancer Therapy, Mimic, Transcription 45 factor, down regulation of gene expression. 46 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.08.083972; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 2 INTRODUCTION 3 Cancer is one of the most prevalent pandemics all over the world causing millions of 4 deaths every year (1-2). At global level, lung cancer, breast cancer and colon cancer are leading 5 cancer types in terms of incidences and mortality rate. Besides conventional anticancer therapy, 6 there are therapeutic avenues to target molecular distinctiveness within the tumors including 7 genetic, transcriptional and microenvironmental heterogeneity (3-9). 8 In essence, genetic and transcriptional heterogeneity are one of key factors that maintain 9 the pro-tumor microenvironment (10-13). Among various driving factors as oncoproteins, 10 transcription factors are large proportions of proteins that bind DNA helix at specific regulatory 11 response elements in order to modulate the expression of a set of genes that contribute towards 12 distinctive features of cancer cells including growth and proliferation (14-18). 13 Transcription factors are known to achieve gain of function that allow the cancer cells to 14 allow the abnormal expression of a set of genes dedicated to hallmarks of tumor including 15 uncontrolled proliferation (3-6, 19-25). In a pool of oncogenic transcription factors, AP-1 16 transcription factor complex consists of c-Fos and c-Jun heterodimers having similar sequence 17 and structure with a bZip protein which helps the AP-1 complex to bind upon specific heptamer 18 consensus nucleotide sequence 5`TGAGTCA3` (AP-1 site) (3, 10-13). This heptamer is also 19 referred to as the 12-O-Tetradecanoylphorbol-13-acetate (TPA) response element. 20 In recent, blockade of AP-1 transcriptional complex is perceived as a promising option to 21 reduce the uncontrollable growth and proliferation of cancer cells by using small molecular 22 inhibitors and peptide mimetics (14-25). These small molecule inhibitors are suggested to bind 23 precisely with the TPA response element of target genes by non-covalent forces such as the 24 coulombic force, vander-waals interactions, and hydrogen bonding and that may be responsible 25 for their anti-cancer properties. (19-25). Evaluation of these molecular inhibitors are evaluated 26 by various approaches including in vitro transcriptional assay and molecular docking 27 computational approach (15-26). 28 There are limited findings that support the use of small molecular inhibitor such as nor- 29 dihydroguaiaretic acid (NDGA), curcumin and tripeptides against the key oncogenic 30 transcriptional complex such as AP-1, FOXO1, MYC and hypoxia inducing factor-3 (14-28). In 31 fact, chemical inhibition of AP-1 transcriptional complex has shown encouraging evidence on 32 retardation of proliferative potential of cancer cells during in vitro and in vivo experiments (29- 33 37). 34 In search of potential small molecular inhibitors and tripeptides as anticancer drugs, 35 limited data indicate that biological fluids such as plasma, urine, milk from human and ruminants 36 contains tripeptides and other metabolites that are shown to display biological properties such as 37 DNA destabilizing and chelation activity, anti-inflammatory and modulation of cellular growth 38 and proliferation (38-46). Among various small molecules, tripeptides are suggested to be 39 transported across the cell membrane by the help of peptide transporters, endocytosis and 40 membrane permeation (47-54). We have also shown that goat urine derived metabolites and 41 tripeptides enriched fraction displayed DNA metabolizing and anti-proliferative activity (45-46). 42 Based on the existing thrust to explore AP-1 transcriptional complex inhibitors, we 43 propose to test the ability of novel tripeptides displaying anti-proliferative effects as a potential 44 inhibitor of AP-1 transcriptional complex. 45 46 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.08.083972; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 2 MATERIALS AND METHODS 3 Materials 4 Cell culture reagents were procured from Himedia India Pvt. Ltd. and Invitrogen India Pvt. 5 Ltd. The HCT-116 cells were obtained from the National Centre For Cell Science (NCCS), Pune, 6 India. Plasmid DNA pBR322, DMSO, acrylamide and other chemicals were of molecular 7 biology grade and these chemicals were purchased from Himedia India Pvt. Ltd and Merck India 8 Pvt. Ltd. 9 Cell Line Maintenance 10 The HCT-116 cells were cultured and maintained in DMEM (Dulbecco’s Modified 11 Eagles Medium) (Himedia) with high glucose supplemented with 10% heat-inactivated 12 FBS/penicillin (100 units/ml)/streptomycin (100 µg/ml) at 37°C in a humidified 5% CO2 13 incubator. The passaging number of HCT-116 cells was maintained below twenty. 14 In Vitro DNA metabolizing assay 15 Goat urine DMSO fraction (GUDF) enriched with tripeptides was used for the evaluation of 16 DNA metabolizing effects by in vitro reaction.