Fragment screening for stabilizers and binders to human DJ-1
Oh, Xu Xuan
Date of Issue2016-08-04
School of Biological Sciences
Parkinson’s disease (PD) is a chronic progressive neurodegenerative disease that is on the rise. Currently, treatments are only available to alleviate the span of symptoms but not to cure the morbidity. This project focuses on finding ligands to increase the stability of DJ1 where its anti-oxidant properties help to reduce the rate of dopaminergic neurons degeneration, delaying onset of PD. The binding of fragments to DJ1 constructs are screened using biophysical techniques. ThermoFluor assay (DSF) is used to screen the fragment libraries where five ligands namely CPD1, CPD2, CPD3, CPD4 and CPD5 were identified to confer stability to DJ1. The assays were performed under control conditions and oxidative conditions from 0x to 20x hydrogen peroxide (H2O2) molarity in excess. In the presence of CPD1, DJ1 was observed to have a positive melting temperature (ΔTm) shift in oxidised DJ1, showing an enhancement of DJ1 stability under oxidative conditions. Isothermal titration calorimetry (ITC) conducted between DJ1 and CPD5 shows evidence of high binding affinity with DJ1. Experiments performed with CPD1 run need to be validated. To learn about the binding sites for the fragments in DJ1, hanging-drop crystallization is attempted and suitable cultured crystals will undergo X-ray crystallography analysis.
DRNTU::Science::Biological sciences::Human anatomy and physiology::Neurobiology