The probes should allow researchers to study the progression of tumours in live animals and how the activity of the cysteine cathepsin proteases which are more active in some cancers as well as several other diseases. Cysteine protease enzymes degrade polypeptides by cleaving peptide bonds and their altered activity has been implicated in the onset of diseases such as cancer, arteriosclerosis, inflammation and Alzheimer's disease. This latest research from Stanford University, US, published in an advance online issue of the journal Nature Chemical Biology discusses the development and testing of the 'activity based probes' (ABP) that are normally invisible to the naked eye, but emit near-infrared (NIR) light on binding to the protease enzyme. Due to its long wavelength NIR light passes through the skin more readily than normal light making it detectable with infra red cameras or film. "In general, an ABP is comprised of an active 'warhead' that covalently binds to a target using an enzyme-catalysed chemical reaction. This reactive group is linked to an element that that confers specificity and directs binding to the target," write the researchers. "The final key element is a reporter tag that allows probe-labelled proteins to be directly visualised or purified from a complex mixture." In vivo imaging of mice tumour models injected with the dye showed that the probes circulated throughout the animal and very rapidly accumulated in cancerous growths showing "virtually no background labelling". "Unlike other enzyme-targeting molecules, it's very specific, sticks to where it binds and does it all very rapidly - in 30 minutes or less," said Dr Matthew Bogyo. The researchers were then able to continue and study the effects of small-molecule protease inhibitors to confirm that the signals we due to protease activity and that the probes have value for monitoring target inhibition. The researchers found that the specific signals from tumours were reduced after treatment with a small molecule dipeptide vinyl sulphone protease inhibitor by between 60 and 80 per cent. The tumour samples were then analysed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) to confirm the signal losses. The tumour samples indicated that labelling of specific cysteine cathepsins was reduced in treated tissues by 50 to 80 per cent. "We went one step beyond just telling if the enzymes are there. We can answer the question, 'Are they active?' That's important because an accumulation of inactive enzymes doesn't necessarily indicate disease," said Dr Bogyo. "[This technique] allows you to see exactly where enzymes are active within living animals."