Work performed since the beginning of the project
The experiments on MET, hypoxia and tumour invasion (Participant 1) have led to the engineering of tumour cell lines in which oxygen availability can be controlled by expression of an oxygen transporter (myoglobin) or a constitutively active mutant of HIF-< and have shown that tumours formed in vivo by myoglobin-expressing cells contained fewer hypoxic regions and display a more differentiated phenotype.
The studies on the cooperation of the MET, WNT and chemokine pathways in metastasis (Participant 2) have built on a genetic strategy using mice. Mice expressing a constitutively active ®-catenin mutant have been crossed with a transgenic line over-expressing HGF/SF. The compound line thus obtained expressed tumours never observed in the single mutants, demonstrating that the MET and WNT pathways strongly cooperate in breast cancer progression.
For the second objective of SFMET, namely a high-resolution structure of the HGF/SFMET complex (Participant 3), several complexes of HGF/SF with soluble fragments of the MET receptor have been characterised and one of these has been successfully crystallised yielding diffraction at 8Å resolution.
Considerable progress has also been made on the third objective, namely the development of novel MET inhibitors for cancer therapy. The work by Participant 4 employed protein engineering in order to convert a fragment of HGF/SF known as NK1 - a partial agonist – into a receptor antagonist. NK1 mutants have been identified that retain MET binding but fail to activate the receptor and crystals of two of these mutants have been obtained.
In a parallel approach (Participant 5), chemical libraries have been screened for compounds binding MET or HGF/SF. The screens have successfully identified binders with affinities in the micromolar range, suitable for fragment assembly or classic medicinal chemistry. In order to define the binding of these molecules to their protein targets, a complete NMR assignment of NK1 has also been successfully produced.
Finally, the work by Participant 6 has led to extensive progress on novel inhibitors of SHP-2, a critical downstream effector of MET signalling. New compounds have been identified that block SHP-2 with remarkable specificity and parallel medicinal chemistry studies on the SHP-2 inhibitor first isolated have led to marked improvements in its potency and specificity.