European Commission logo
English English
CORDIS - EU research results
Content archived on 2024-06-16

Structural & dynamical characterization of the pro-metastatic enzyme heparanase for the designing of novel inhibitors

Final Activity Report Summary - ANTICANCER NMR (Structural & Dynamical Characterization of the Pro-Metastatic Enzyme Heparanase for the Designing of Novel Inhibitors)

The focus of the project is the understanding of the biological functions and the structural characterisation of heparanase. Heparanase is an endo--D-glucosidase capable of specifically degrading heparan sulfate, one of the extracellular matrix (ECM) components. This activity is associated with the metastatic potential of tumour cells, because these cells require the ability to degrade the ECM. Heparanase is thus an attractive target for the identification of small compounds which will be able to inhibit metastatic heparanase, in order to design new anticancer drugs. NMR spectroscopy has become a leading technique to study biomolecules. The major limitation of NMR arises from the increase of transverse relaxation rates which prevents the investigation of molecules with large molecular weights. Heparanase is initially expressed as a 543 aminoacid polypeptide (65 KDa). For this reason, an important molecular biological effort has been undertaken to build smaller constructs of heparanase whose structural analysis by NMR will be affordable.

During this year, several different constructs have been designed and cloned, and their expression and purification conditions optimised. These constructs include the 'core' or catalytic subunit of the protein as well as the complete form of adult heparanase and also the renal-fetal isoform. An in depth study on the best conditions for the preparation of the NMR sample was necessary due to the low solubility of the different constructs in water. However, we have been able to solubilise the protein by the use of non-denaturant detergents and concentrate it in order to obtain an adequate sample for NMR measurements.

We have obtained good quality 1H,15N-HSQC spectra for all the adult constructs, but we have focus our studies on the 'core' because it is the smallest construct (29 KDa) and is contained in all forms of the construct. We have been able to obtain, mono- (15N), double (13C, 15N) and triple-labelled (2H, 13C, 15N) samples. After performing all the NMR experiments necessary for the backbone sequence specific assignment, we are now in the process of analysing them. Up to now, we have identified 20 % of the amino acids.

The catalytic centre of the protein has also been identified and thus far we have been able to perform preliminary screening studies with some known inhibitors of Heparanase. The results obtained from these studies will address the primary hypothesis of the study, that is, the use of Heparanase as a target for drug discovery.

In addition to the work performed on the Heparanase project, I have also been developing the group's metabonomics research program and I am involved in a number of projects in this area, the main one involving the study of cirrhosis. In this project, NMR-based metabonomics is being applied to determine the underlying metabolic pathways involved in the degeneration of cirrhosis to hepatic encephalopathy. The first step of this project has consisted of examining blood serum metabolite profiles of patients suffering from cirrhosis and the first stage of encephalopathy. Using multivariate statistics methods we have been able to separate the three sample groups (controls, cirrhotic and minimal hepatic encephalopathy) and identify some of the discriminating metabolites. In another project, we have also applied metabonomics for the classification of blood serum samples of healthy controls with those collected from patients suffering B cell chronic lymphocytic leukaemia based on differences in their blood metabolic profiles. Similar applications of metabonomics are also being employed where we are investigating the mechanisms controlling Embryonic Stem Cell (ESC) fate.

I am also working in the dynamical and structural characterisation of Tah1. Tah1 is a tetratricopeptide repeat (TPR) protein and it has been shown to bind the C-terminal end of Hsp90, which is an ubiquitous molecular chaperone found in eubacteria and all branches of eukaryotes. Finally, I am co-supervisor to Guillermo Badenes, PhD student whose thesis aims to solve the structure of the hemopexin domain of the human membrane type 1 metalloproteinase, MT1-MMP or MMP-14.