1. Pyruvate Dehydrogenase Kinase 4: A potential drug target in non insulin dependent diabetes mellitus Thandeka Khoza

2. © CSIR 2007 www.csir.co.za Background Type 2 diabetes mellitus (T2DM) affects 6 % of modern society Expected to grow by 6 % annually Insulin resistance and obesity risk factors for development of T2DM During insulin resistance, - Cells that are involved in glucose clearance are unable to respond to insulin signaling - High levels of glucose in blood

3. © CSIR 2007 www.csir.co.za Pyruvate Pyruvate + CoA + NAD + Acetyl-CoA + NADH + CO 2 PDC Acetyl-CoA Lipid biosynthesis cytosol mitochondria Role of PDC in glucose metabolism Gluconeogenesis Glycolysis Gluco se PDC: pyruvate dehydrogenase complex

4. © CSIR 2007 www.csir.co.za Regulation of PDC PDC: Pyruvate dehydrogenase complex PDK: Pyruvate dehydrogenase kinase PDP: Pyruvate dehydrogenase phosphatase

5. © CSIR 2007 www.csir.co.za PDK4 and its link to T2DM PDK4 is believed to be the isoform involved in diabetes - High expression in tissues involved with metabolic clearance of glucose - PDK4 is highly expressed in diabetic conditions - It shows high activity towards phosphorylation of PDC site 2 this site is believed to be important in diabetes

6. © CSIR 2007 www.csir.co.za Regulation of PDK PDK Inhibits Activates NAD + Pyruvat e ADP CoA NADH Acetyl- CoA Pyruvate + CoA + NAD + Acetyl-CoA + NADH + CO 2 PDC PDC: Pyruvate dehydrogenase complex PDK: Pyruvate dehydrogenase kinase

7. © CSIR 2007 www.csir.co.za Classification of PDK

8. © CSIR 2007 www.csir.co.za Inhibitors of PDK Sodium Dichloroacetate (DCA) DCA (sodium dichloroacetate) - Only inhibitor to reach clinical trials - Improves diabetic condition - Not ideal for long-term use due to toxicity of its metabolite - Other drugs such as ATP analogues and Lactones are still at research level So the hunt is on to find PDK inhibitors that can be used in the treatment of T2DM

9. © CSIR 2007 www.csir.co.za Aim To examine the interaction of PDK4 with its ligands (ADP and ATP)

10. Project Outline Structural biology Biochemistry Expression of rPDK4 Investigation of ADP binding site, potentially a worthwhile target site for inhibition of rPDK4 activity? Identification of suitable template Indentification of aa's interacting with ADP / ATP Binding of ADP and ATP to mutated rPDK4 Homology modelling Affinity purification of rPDK4 Enzyme characterization – Interaction with ATP / ADP Site directed mutagenesis

11. © CSIR 2007 www.csir.co.za Homology modelling of rPDK4 rPDK2 was choosen as template for homology modelling of rPDK4 - rPDK2 and rPDK4 have similar physiological function - rPDK2 and rPDK4 have 70 % similarity - Structure of rPDK2 has been experimentally solved rPDK4 was modelled using Swiss-Model R ibbon presentation of modelled rPDK4 dimer. α-Helices are red, β-strands are yellow, and the loop regions are pink.

12. © CSIR 2007 www.csir.co.za Interaction of PDK and ADP rPDK2 (PDB code- 1JM6) rPDK4

13. © CSIR 2007 www.csir.co.za Structural analysis of rPDK4 Has high primary amino acid sequence similarity to that of rPDK2 The ADP binding site of rPDK2 is homologous to that of rPDK4 The model suggests that the mechanism of ADP binding to rPDK2 and rPDK4 is similar

14. © CSIR 2007 www.csir.co.za Methodology Purification of rPDK4 Expression of rPDK4 Characterization Binding of ADP to PDK

15. © CSIR 2007 www.csir.co.za Gel filtration and size determination 50 kDa 10 % SDS PAGE gel Western blot analysis Retention time of 116 min, molecular weight was estimated to be 96.76 kDa rPDK4 protein was sucessfully expressed and partially purified using affinity chomatography

16. © CSIR 2007 www.csir.co.za Characterization of rPDK4 by Tryptophan Fluorescence PDB code – 2BU2 ATP – red Tryptophan - blue PDK2 Trp - 383

17. © CSIR 2007 www.csir.co.za Fluorescence Quenching of PDK by ATP at 350 nm

18. © CSIR 2007 www.csir.co.za Fluorescence Quenching of PDK by ADP at 350 nm

19. Future Work Structural biology Biochemistry Expression of rPDK4 Investigation of ADP binding site, potentially a worthwhile target site for inhibition of rPDK4 activity Identification of suitable template Identification of aa's interacting with ADP / ATP Binding of ADP and ATP to mutated rPDK4 Homology modelling Affinity purification of rPDK4 Enzyme characterization – Interaction with ATP / ADP Site directed mutagenesis

20. Acknowledgements Prof. Heini Dirr, University of the Witwatersrand Dr Lungile Shoba-Zikhali, CSIR Biosciences CSIR Biosciences Structural Biology CSIR Biosciences Enzyme Technologies