"Small molecule screen to identify inhibitors of DUX4-mediated toxicity, therapeutic approach for FSHD"

Bosnakovski, Darko (2012) "Small molecule screen to identify inhibitors of DUX4-mediated toxicity, therapeutic approach for FSHD". [Project] (In Press)

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Abstract

Aim 1. To narrow our focus to the most promising direct DUX4 inhibitors. From the current 82 selected compounds which rescue DUX4 toxicity, we will narrow down the list to direct DUX4 inhibitors by means of additional secondary screens. The first screen will be an analysis of MyoD expression in DUX4 induced and chemical compound treated cells. Levels of MyoD will be determined by qRT-PCR after 24 and 72 hours of incubation. To analyze the effect of selected compounds to DUX4 protein, meticulous secondary screens based on immunofluoroscence will be performed. DUX4 expressing myoblasts will be treated with our cherry-picked compounds and levels, stability and localization of the DUX4 protein will be evaluated. Analyzed compounds may have no effect on DUX4 protein levels, may reduce levels by decreasing the stability of the protein, or may alter cellular localization such that less DUX4 accumulates in the nucleus. After these two screens, we hope to have a list of 20 potent chemicals which will be evaluated separately for purity, solubility, pharmacological activity and DUX4 binding affinity. For the compounds that bind DUX4 directly, we will generate 5-10 derivative compounds to confirm structure activity relationships and hopefully achieve higher activity. The purpose of this aim is to find chemicals that bind directly to the Dux4 protein, are highly soluble, are non-reactive, and are active at low concentration (100 nM). Aim 2. To evaluate effectiveness of DUX4 inhibition We will analyze DUX4 target genes in iC2C12-DUX4 rescued cells. Using qRT-PCR we will evaluate a panel of DUX4-downstream genes, including myogenic transcription factors (MRFs), oxidative stress-related genes, and key toxicity associated downstream targets. To confirm the effectiveness of DUX4, similar transcription assays will be performed on i3T3-DUX4 cells. A global gene expression array will be carried out for each compound. This will allow us to evaluate the completeness of inhibition. As a functional in vitro assay, we will analyze the level of myogenic differentiation in DUX4 induced myoblasts. We will check for the ability of the compounds to revert the phenotype of FSHD myoblast. Treated FSHD myoblasts will be evaluated for morphological abnormalities, susceptibility to oxidative stress, integrity of MyoD expression and MyoD downstream targets, and differentiation capacity. Finally, we will determinate whether our compounds inactivate DUX4 protein or block its transcription/translation in FSHD primary myoblasts. Aim 3. To analyze pharmacokinetic and pharmacodynamic properties of the selected compounds. Pharmacokinetic and pharmacodynamic properties of the selected compounds will be analyzed by predicting metabolism using a human hepatocyte microsome assay, by measuring concentration over time in plasma by high-end mass spectrometry, and by toxicity studies in mice. Furthermore, LD10 and LD50 will be determined.

Item Type: Project
Subjects: Medical and Health Sciences > Basic medicine
Divisions: Faculty of Medical Science
Depositing User: Marijana Kroteva
Date Deposited: 27 Nov 2012 14:14
Last Modified: 03 Dec 2012 13:36
URI: http://eprints.ugd.edu.mk/id/eprint/2249

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