Novel antiproliferative tripeptides block AP-1 transcriptional complex by in silico approach

Abstract

BACKGROUNDThe complexity and heterogeneity at genetic, epigenetic and microenvironment levels are key attributes of tumors. Genetic heterogeneity encompasses one of key factors at transcriptional gene regulation that promote abnormal proliferation, invasiveness and metastasis. Among various key pro-tumor transcriptional complexes, activating protein-1 (AP-1) transcriptional complex controls the transcriptional expression of key oncogenes in cancer cells. Therefore, an avenue to search for a chemical inhibition approach of the AP-1 transcriptional complex is warranted in cancer therapeutics.METHODSTo achieve chemical inhibition of AP-1 transcriptional complex, we report novel tripeptides identified from the goat urine DMSO fraction as potential agents that bind to AP-1 responsive TPA element and heterodimer c-Jun:c-Fos. Novel tripeptides enriched GUDF were tested against DNA substrates to assess DNA metabolizing activity. Further, Novel tripeptides enriched GUDF were treated upon HCT-116 cells to estimate the nature of tripeptides entered into the intracellular compartment of HCT-116 cells. Here, we report on a novel methodology that employ VTGE assisted intracellular metabolite purification and is analyzed with the help of LC-HRMS technique. Post purification of intracellular metabolites that included tripeptides of GUDF, these tripeptides from DMSO and GUDF treated HCT-116 cells were subjected to molecular docking and ligand-DNA:AP-1 (PDB ID: 1FOS) interaction study by using bioinformatics tools AutoDock Vina and PyMol.RESULTSGUDF enriched with tripeptides and other metabolites show appreciable instability of DNA substrates plasmid and genomic DNA to an extent of 90%. Interestingly, LC-HRMS analysis of intracellular metabolite profiling of GUDF treated HCT-116 cells reveal the appreciable abundance of tripeptides Glu-Glu-Arg, Gly-Arg-Pro, Gln-Lys-Arg, Glu-Glu-Lys, Trp-Trp-Val. On the other hand, DMSO treated HCT-116 cells show the presence of Ser-Trp-Lys, Glu-Glu-Gln, Glu-Glu-Lys, Ser-Leu-Ser. Interestingly, GUDF treated HCT-116 cells show inhibition of proliferation by more than 70%. Among the identified intracellular tripeptides, Glu-Glu-Arg (9.1 Kcal/Mol), Gly-Arg-Pro (8.8 Kcal/Mol), and Gln-Lys-Arg (6.8) show a precise and strong binding to heptameric TPA response element 5’ TGAGTCA 3’ and key amino acid residue within the AP-1 transcriptional complex.CONCLUSIONIn summary, this study suggests the potential of novel tripeptides, those are reported from GUDF intracellularly in HCT-116 cells to destabilize the AP-1 transcriptional complex. Data indicate that cellular arrest in HCT-116 cells treated by GUDF is well supported by the molecular docking observations that destabilization of AP-1 complex is linked to reduced growth and proliferation.

Predicted Role of Acetyl-CoA Synthetase and HAT p300 in Extracellular Lactate Mediated Lactylation in the Tumor: In vitro and In silico Models

“…The extracellular conditioned medium of MCF-7 breast cancer cells treated by DMSO and cow urine DMSO fraction (CUDF) (50 µg/ml) final concentration was collected as per the previously published procedure (41)(42)(43). Then, 750 µl of extracellular conditioned medium was mixed with 4X loading buffer (0.5 M Tris, pH 6.8, and Glycerol).…”

“…Then, 750 µl of extracellular conditioned medium was mixed with 4X loading buffer (0.5 M Tris, pH 6.8, and Glycerol). Next, the conditioned medium along with the loading buffer was loaded on the vertical tube gel electrophoresis (VTGE) purification system with a matrix of 15% polyacrylamide gel (acrylamide: bisacrylamide, 30:1) (42)(43). The fractionated extracellular metabolites were collected in the running buffer (96 mM glycine, pH 8.3).…”

“…The fractionated extracellular metabolites were collected in the running buffer (96 mM glycine, pH 8.3). The detailed procedure was adopted from previously published in-house VTGE-assisted purification of metabolites (42)(43). Furthermore, LC-HRMS analyses of VTGE-purified extracellular metabolite elutes were performed by Agilent TOF/Q-TOF Mass Spectrometer station Dual AJS ESI ion source.…”

“…During LC separation, RPC18 Hypersil GOLD C18 100 x 2.1 mm-3 µm and mobile phase of 100% Water (0.1% FA in water) and 100% Acetonitrile (90% ACN +10% H2O+ 0.1% FA) were used in the proportion of 95% and 5% (40). Mass spectrometry was performed in a positive mode and analyzed as per the procedure adopted from previously reported methodology (43).…”

“…In this regard, we have attempted to detect the levels of lactate and lactyl-CoA at the intracellular and extracellular levels of breast cancer cells treated by DMSO and anticancer drug compositions enriched with free fatty acids and tripeptides (41)(42)(43). The use of anticancer drug composition CUDF is taken as one of the candidate drug models to see the difference in the detection of lactate and lactyl-CoA compared to the DMSO control.…”