Identifying biologically relevant, non-teratogenic analogs of Thalidomide to understand and combat the inflammatory response and its disorders

Supervisors: Neil Vargesson, Megan Davey

Project description:

Thalidomide notoriously caused birth defects to thousands of children in the 1960's where it was used to treat morning sickness. Thalidomide is now used to effectively treat Leprosy and Multiple Myeloma. Tragically, a new generation of thalidomide-damaged children are now being observed in Brazil where the drug is used to treat Leprosy but is not controlled as it is in the UK and USA. This project is focused on identifying and screening new analogs of thalidomide that retain the clinically and biologically relevant aspects but not the side-effects.

Using our published assays using Zebrafish and Chicken embryos as well as in-vitro cell culture assays (Beedie et al., 2015; Beedie et al., 2016), together with fluorescently labelled tissue specific transgenic Chicken embryos (Davey Lab) over 35 novel thalidomide analogs and breakdown products will be screened to identify their actions and primarily identify those with anti-inflammatory and non-teratogenic actions. The transgenic chicken embryos will confirm the tissue specific actions of the compounds and shed light on how the compounds affect tissues. Such identified analogs will then be screened in human cancer and inflammatory tissue culture assays to confirm their actions. Additionally, potent anti-inflammatory analogs identified may also be screened in animal models of arthritis to confirm their action/s. The student will also analyse molecular pathways related to the inflammatory response in embryos and determine molecular targets that the analogs target.

Methodology and Training:

Thalidomide analog’s will be applied to chicken embryos (wildtype and transgenic) around day 3 of development, when most of the body system is developing. Compounds will also be screened in Zebrafish embryos to study effects on blood vessel formation and on the inflammatory response, using two transgenic reporter lines. 1. Fli-1EGFP –a fish line where the endothelial cells are tagged with enhances green fluorescence protein (EGFP) allowing the vessels to be observed. 2. MPO-GFP –a fish line where neutrophils are tagged with GFP and in response to an injury the neutrophils are activated to the wound site to help heal the wound. The PhD student will identify compounds that are anti-inflammatory but not antiangiogenic (Beedie et al., 2015; Beedie et al., 2016; Therapontos et al., 2009). The student will also look at TNFalpha levels following ELISA analysis on in-vitro cell cultures following analog exposure, a major molecule involved in induction of the inflammatory response to injury or stimulus to determine the relative potency of the compounds.

Analogs with anti-inflammatory only (and no teratogenic actions) will also be tested in human cell lines of cancer and animal models of inflammation (ie: arthritis) to confirm their actions. Analogs with teratogenic actions will be analysed further in transgenic chicken embryos to study tissue changes following drug exposure and determine how the drugs causes defect; the developing limb will be the focus.

Year 1 and 2 will involve learning embryology and carrying out the analog screening. Year 3 will involve in-vitro cell culture analyses and in-vivo animal models of inflammation and teratogenesis.