Using novel drug discovery platforms to inhibit ENPP enzymes during the calcification process

Supervisors: Scott WebsterVicky MacRaePatrick Hadoke

Project Description:

Heart disease and strokes affect about one person in three and are in part caused by arteries and valves becoming stiffened with calcium deposits (1). However, how this vascular calcification occurs is not fully known.

Recent studies by our research group (2) have investigated how the muscle cells found normally in the walls of arteries partially transform themselves into bone cells and lay down deposits of calcium salts similar to the substances found in the skeleton. The overall result is a thickening and stiffening of the artery wall and a higher risk for heart disease.

This project will study valve development and function and will utilise in vitro culture models of vascular cells, including valve interstitial cells (VICs). Specifically, the project will investigate two enzymes within the ectonucleotide   pyrophosphatase /phosphodiesterase family, ENPP1 (3) and ENPP2 (also known as Autotaxin), which have been implicated in VIC calcification. Prof Webster’s laboratory have been pursuing a therapeutic discovery programme focused on the ENPP enzymes, identifying potent small molecule inhibitors of ENPP2 (and ENPP6) and generating an ENPP isozyme screening platform. This studentship will extend this programme to investigate ENPP1 and 2 inhibition by generating a target validation package for these enzymes as crucial tools for discovering the mechanisms underpinning VIC calcification. Studies will involve the assessment of calcification in vitro and in vivo through the quantification of calcium and phosphate deposition using biochemical assays and fluorescent probes. We will also use our models of vascular calcification to investigate the role of different pathways that may be regulated by ENPP1 and ENPP2 during the calcification process; for example Wnt signalling and autophagy. In addition, RNAseq with subsequent informatics analysis will be undertaken to identify novel underpinning mechanisms. Techniques used will include histology, qPCR, western blotting, immunofluorescent staining and siRNA knockdown. 

Training: The supervisory team of this project merges expertise in drug discovery (SW), cardiovascular biology (VEM, PH), calcification biology (VEM, PH), and pharmacology (SW). Training will be primarily through practical application of research techniques, analytical methods and study skills in the supervisors’ laboratories, as well as The Centre for Cardiovascular Science’s core imaging facility, which houses the drug discovery team. Skills that will be acquired include cell and molecular biology techniques, enzyme assay, protein expression & purification, microscopy, cell culture and in vivo techniques. Students are also encouraged to attend monthly Research Workshops dedicated to student and post-doctoral presentations. 

Anticipated outputs: The student will be encouraged to present their work at International and European scientific conferences. Past students of the supervisors’ have typically published at least 3 scientific papers during their studentships, and have subsequently secured high profile post-doctoral research positions in the UK, Europe and USA.

1.    Zhu D, Mackenzie NC, Farquharson C, MacRae VE 2012 Mechanisms and clinical consequences of vascular calcification. Front Endocrinol. 3:95.
2.    Zhu D, Hadoke PW, Wu J, Vesey AT, Lerman DA, Dweck MR, Newby DE, Smith LB,  MacRae VE 2016 Ablation of the androgen receptor from vascular smooth muscle cells demonstrates a role for testosterone in vascular calcification. Sci Rep 6:24807.
3. Mackenzie NC, Huesa C, Rutsch F, MacRae VE 2012 New insights into NPP1 function: lessons from clinical and animal studies. Bone. 51:961-8.
4. Mole DJ, Webster SP, Uings I, Zheng X, Binnie M, Wilson K, Hutchinson JP, Mirguet O, Walker A, Beaufils B, Ancellin N, Trottet L, Bénéton V, Mowat CG, Wilkinson M, Rowland P, Haslam C, McBride A, Homer NZ, Baily JE, Sharp MG, Garden OJ, Hughes J, Howie SE, Holmes DS, Liddle J, Iredale JP. (2016) Inhibition of kynurenine-3-monooxygenase activity protects against multiple organ failure in rodent models of severe acute pancreatitis. Nat Med, 22: 202-209.

If you wish to apply for this project, please check this link and send your application to this email.