Identification Of Potential Cellular Targets Research Articles

Abstract 3785: Identification of potential cellular targets for Epstein-Barr virus encoded microRNAs miR-BART7 and miR-BART9 by in silico analysis

Abstract The Epstein-Barr virus (EBV), formally designated human herpesvirus 4 (HHV-4), is an ubiquitous virus that causes lifelong latent infection in over 90% of the world's human adult population. EBV infection is regarded as carcinogenic to humans and associated with a variety of human cancers, especially endemic Burkitt lymphoma and nasopharyngeal carcinomas. EBV was the first human virus described to encode viral microRNAs (miRs), but the activity of EBV-encoded miRs in carcinogenesis remains to be fully elucidated. In this study we sought to identify novel potential targets for EBV-encoded miR-BART7 and miR-BART9 using in silicoanalysis. Mature sequences for EBV-miR-BARTs 7-3p, 7-5p, 9-3p, and 9-5p were retrieved from miRBase (http://www.mirbase.org, version 22) and 3’-UTRs from Homo sapiens were obtained from Ensembl (https://www.ensembl.org, version 92). RNAhybrid, miRanda, and PITA softwares were used for target predictions of miR-binding sites within 3’-UTRs of human mRNAs. Overlapped targets were collected using a custom Python script in such a way that only genes predicted by all the three algorithms were selected and used for gene-enrichment pathway analysis using the Cytoscape with the Reactome FI database plug-in (version 3.6.0). Each gene in a given pathway was counted as one hit using another custom Phyton script, and results were considered significant when P-value <0.05. As result, high-scoring predicted targets for EBV-miR-BARTs 7 and 9 were found to affect pathways involved in immune responses, gene expression, metabolism of proteins, and signal transduction. By targeting key tyrosine kinases pathways, such as epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), and vascular endothelial growth factor (VEGF), EBV-miR-BART7-3p was predicted to play a significant role in cell proliferation, cell cycle progression, and tumorigenesis. On the other hand, EBV- miR-BART7-5p was found to regulate adaptive immune system antigen presentation via MHC class II, cytokine signaling, and growth hormone receptor signaling. EBV-miR-BART9-3p was found as a relevant regulator for post-translational modification of proteins, WNT/β-catenin signaling, and pre-mRNA processing, while EBV-miR-BART9-5p could exert a role on signaling mediated by Rho GTPases, G protein- coupled receptors (GPCRs), gene silencing by small RNAs and RNA polymerase II transcription pathways. In conclusion, the results indicate pathways and genes that may be substantially targeted by EBV-miR- BARTs 7 and 9 and deserve further experimental investigation to disclose possible roles of EBV miRs on pathogenesis of cancers associated with viral infection. This work was supported by Sao Paulo Research Foundation (FAPESP) under grants 17/20352-0, 17/22312-5 and 17/23393-9. Citation Format: Brunno F. Caetano, Bárbara G. Müller-Coan, Rafael L. Coan, Bruno E. Fantinatti, Deilson E. de Oliveira. Identification of potential cellular targets for Epstein-Barr virus encoded microRNAs miR-BART7 and miR-BART9 by in silico analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3785.

Target identification of biologically active small molecules via in situ methods

The identification of potential cellular targets of small molecules is important in biomedical research and drug discovery, but has been challenging due to a lack of proteome-based methods that enable direct investigation of small molecule-protein interaction in live cells. This review summarizes some of the recent advances in target identification of bioactive molecules (including drugs and natural products) using in situ methods for cell-based proteome profiling of potential on and off targets.

Cell‐Based Proteome Profiling Using an Affinity‐Based Probe (AfBP) Derived from 3‐Deazaneplanocin A (DzNep)

3-Deazaneplanocin A (DzNep), a global histone methylation inhibitor, has attracted significant interest in epigenetic research in recent years. The molecular mechanism of action and the cellular off-targets of DzNep, however, are still not well-understood. Our aim was to develop novel DzNep-derived small-molecule probes suitable to be used in live mammalian cells for identification of potential cellular targets of DzNep under physiologically relevant settings. In the current study, we have successfully designed, synthesized, and tested one such probe, called DZ-1. DZ-1 is a 'clickable' affinity-based probe (AfBP) derived from DzNep with minimal structural modifications. The probe was found to be highly cell-permeable, and possessed similar anti-apoptotic activities as DzNep in MCF-7 mammalian cells. Two additional control probes were made as negative labeling/pull-down probes in order to minimize false identification of background proteins due to unavoidable, intrinsic nonspecific photo-crosslinking reactions. All three probes were subsequently used for in-situ proteome profiling in live mammalian cells, followed by large-scale pull-down/LC-MS/MS analysis for identification of potential cellular protein targets that might interact with DzNep in native cellular environments. Our LC-MS/MS results revealed some highly enriched proteins that had not been reported as potential DzNep targets. These proteins might constitute unknown cellular off-targets of DzNep. Though further validation experiments are needed in order to unequivocally confirm these off-targets, our findings shed new light on the future use of DzNep as a validated chemical probe for epigenetic research and as a potential drug candidate for cancer therapy.

Proteomic profiling and potential cellular target identification of K11777, a clinical cysteine protease inhibitor, in Trypanosoma brucei

We report herein the design, synthesis and application of K11777-derived activity-based probes (ABPs) allowing in situ profiling and identification of potential cellular targets of K11777 in Trypanosoma brucei.

Chemically Induced Cardiomyogenesis of Mouse Embryonic Stem Cells

A transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of alpha-myosine heavy chain (alpha-MHC) promoter (palpha-MHC-EGFP) was used to investigate the effects of (thio)urea and cinchona alkaloid derivatives on cardiomyogenesis. The screening of the compounds yielded cardiomyogenesis inducing substances with good (IV-5, V-4) to very good activities (II-16, IV-8), as determined by a 50 to 80 % increase in the EGFP fluorescence compared to untreated cells. Time-dependent screening approaches in which compounds were added at different developmental stages of the ES cells appeared to be of limited suitability for the identification of potential cellular targets.

Pathology of Pulmonary Arterial Hypertension

The pathological features of pulmonary arterial hypertension are highlighted in this review, which can serve as a guide to diagnosis and identification of potential cellular targets for novel lines of therapy. It also emphasizes the variability of pathological processes found in pulmonary hypertensive lungs collected by the Pulmonary Breakthrough Initiative of the Cardiovascular Medical Research Fund. This initiative is aimed at banking properly phenotyped lung tissue and implementing relevant human lung-based investigations of pulmonary arterial hypertension. This effort has led to an insight of site-specific heterogeneity of relevant pulmonary vascular pathologies and how associated histopathological processes, including interstitial fibrotic and inflammatory findings, may need to be considered in the development of future therapies. We also highlight the pulmonary vascular pathology in schistosomiasis-associated pulmonary arterial hypertension, possibly the most frequent cause of pulmonary hypertension worldwide.

Identification of potential cellular targets of aloisine A by affinity chromatography

Affinity chromatography was used to identify potential cellular targets of aloisine A (7- n-butyl-6-(4′-hydroxyphenyl)- 5H-pyrrolo[2,3 b]pyrazine), a potent inhibitor of cyclin-dependent kinases. This technique is based on the immobilization of the drug on a solid matrix, followed by identification of specifically bound proteins. To this end, both aloisine A and the protein-kinase inactive control N-methyl aloisine, bearing extended linker chains have been synthesized. We present the preparation of such analogues having the triethylene glycol chain at different positions of the molecule, as well as their immobilization on an agarose-based matrix. Affinity chromatography of various biological extracts on the aloisine matrices allowed the identification of both protein kinases and non-kinase proteins as potential cellular targets of aloisine.