Targeting of tissue transglutaminase for functional tumour imaging: Studies on novel assay methods and inhibitors for this enzyme

An increased activity of tissue transglutaminase (TGase 2) in tumours correlates with enhanced invasive potential as well as resistance to chemo- and radiotherapy. Therefore, this enzyme represents an interesting target for the development of PET tracers for functional in vivo imaging of tumours.
One important prerequisite for the identification and characterisation of TGase 2-binding compounds are reliable assay methods to measure the enzymatic activity. For this, a continuous fluorimetric activity assay was established, which allows the detection of the TGase 2-activity through the measurement of an increase in fluorescence. In this context, six novel water-soluble fluorogenic acyl donors containing either 7-hydroxycoumarin or 7-hydroxy-4-methylcoumarin (HMC) as fluorogenic leaving groups were developed and extensively characterised concerning their enzymatic hydrolysis and aminolysis. Within these substrates, the dipeptide Z-Glu(HMC)-Gly-OH exhibits not only the most favourable substrate properties of all compounds in this study but also within the peptidic acyl donors described for TGase 2 so far. In addition to that, a fluorescence anisotropy-based assay method was established where the TGase 2-mediated incorporation of either fluorescein- or rhodamine-conjugated cadaverine into N,N-dimethylcasein is quantified.
For the development of PET tracers for molecular imaging of TGase 2, different approaches are pursued. One of those exploits the use of irreversible inhibitors for this enzyme. Among the TGase 2 inhibitors described in the literature, the recently reported N^α-acyl-N^ε-acryloyl-lysine-4-pyridylpiperazides seem to be most suitable for radiotracer development as these compounds exhibit strong inhibitory potential and selectivity towards TGase 2 as well as favourable pharmacokinetic properties. Hence, derivatives based on this class of compounds that allow the labelling with radionuclides such as fluorine-18 and iodine-124 were prepared and their inhibitory potential towards TGase 2 was evaluated by the two independent assay methods outlined above. The kinetic characterisation of the compounds revealed interesting structure-activity relationships. Particularly, the introduction of iodine into the C-terminal pyridyl moiety resulted in a significantly increased inhibitory potential towards TGase 2 compared to the lead structure. This was further illustrated by investigations on covalent docking of the lysine-derived inhibitors within the catalytic centre of TGase 2 which simultaneously will open strategies for the design of even more potent inhibitors.